https://wiki.tanakalab.org/api.php?action=feedcontributions&user=Sergej.nowoshilow&feedformat=atomTanaka Wiki - User contributions [en]2024-03-29T12:50:32ZUser contributionsMediaWiki 1.35.8https://wiki.tanakalab.org/index.php?title=Axolotl&diff=916Axolotl2020-06-08T07:35:43Z<p>Sergej.nowoshilow: Blanked the page</p>
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<div></div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Overview&diff=915Overview2020-06-08T07:33:20Z<p>Sergej.nowoshilow: /* Axolotl */</p>
<hr />
<div>== Introduction ==<br />
On the pages listed below you can find most common protocols used in the lab as well as a list of lab duties along with the names of the responsible persons. Please, contact the responsible persons if you have any question, suggestions or troubles.<br />
<br />
== Lab duties ==<br />
<ul><br />
=== Chemicals ===<br />
<li>[[Antibiotics|Antibiotics]]</li><br />
<li>[[Benzocaine|Benzocaine]]</li><br />
<li>[[Fibronectin|Fibronectin]]</li><br />
<li>[[Gelatine|Gelatine]]</li><br />
<li>[[Liquid_nitrogen|Liquid nitrogen]]</li><br />
<li>[[Primary_antibodies|Primary antibodies]]</li><br />
<li>[[Secondary_antibodies|Secondary antibodies]]</li><br />
<li>[[Serum_heat_inactivation|Serum heat inactivation]]</li><br />
<br />
=== Hardware ===<br />
<li>[[Autoclave|Autoclave]]</li><br />
<li>[[Cryostat|Cryostat]]</li><br />
<li>[[Dissecting_microscopes|Dissecting microscopes]]</li><br />
<li>[[Leica_confocal_microscope|Leica confocal microscope]]</li><br />
<li>[[Vacuum_pumps|Vacuum pumps]]</li><br />
<li>[[Waterbath|Waterbath]]</li><br />
<li>[[RAMP Test|RAMP Test]]</li><br />
<br />
=== General ===<br />
<li>[[Ordering_common_stuff|Ordering common stuff]]</li><br />
<li>[[Heat_shock_competent_cells|Heat-shock competent cells]]</li><br />
</ul><br />
<br />
== Antibodies ==<br />
=== List of primary antibodies ===<br />
{| class="wikitable sortable"<br />
! Name !! Supplier !! Cat. No !! Host !! Mono/Poly !! Antigen !! class="unsortable" | Contact person !! class="unsortable" | Storage temp !! class="unsortable" | Stock concentration !! class="unsortable" | Conditions for axolotl !! class="unsortable" | Conditions for Xenopus !! class="unsortable" | Conditions for mouse !! class="unsortable" | Conditions for cell culture !! class="unsortable" | Reference<br />
|-<br />
| 12/101 || Developmental Studies Hybridoma Bank || || Mouse || || || Yuka || 4°C || 46 ug/ml Ig || 1:200 || 1:200 || || || || <br />
|-<br />
| 15.3B9 (NOT1) || Developmental Studies Hybridoma Bank || 15.3B9 (NOT1) || Mouse || Monoclonal || notochord marker (Chicken) || Yuka || 4°C || || 21 ug/ml IG || || || || http://dshb.biology.uiowa.edu/notochord-marker || <br />
|-<br />
| 20B4 || Developmental Studies Hybridoma Bank || 20B4 || Mouse || Monoclonal || neural crest cell (Chicken) || Yuka || 4°C || || || || || || http://dshb.biology.uiowa.edu/neural-crest-cells || <br />
|-<br />
| 5D3 || Developmental Studies Hybridoma Bank || 5D3 || Mouse || Monoclonal || Cadherin E (Xenopus) || Yuka || 4°C || || 37 ug/ml IG || || || || http://dshb.biology.uiowa.edu/5D3 || <br />
|-<br />
| 8C8 || Developmental Studies Hybridoma Bank || 8C8 || Mouse || Monoclonal || integrin beta-1/ CD29 (Xenopus) || Yuka || 4°C || || || || || || http://dshb.biology.uiowa.edu/integrin-beta-1_7 || <br />
|-<br />
| Aggrecan || millipore || AB1031 || Rabbit || || || Lidia || || || || Not work, Dim || '1:500 (Lidia) || || || <br />
|-<br />
| Alpha tubulin (DM1a) || MPI || || Mouse || Monoclonal || Chicken alpha tubulin || Takuji || -20°C || || || || || 1:1000 - || || <br />
|-<br />
| b-catenin || Gift from Thomas Kurth || P14L || Rabbit || || Xenopus || Yuka_Aliquot from Tomas Kurth || || || || || || || Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. || <br />
|-<br />
| Bra || || AF2085 || || || || Yuka || -20C || || 0.2ug/ml in PBS || || || || || <br />
|-<br />
| BrdU (Bu20a) || MPI || || Mouse || Monoclonal || BrdU - thymidine analog || Takuji || -20°C || || 1:400 - || || || || || <br />
|-<br />
| BrdU || Antibodies-online || ABIN964591 || Rabbit || Polyclonal || BrdU - thymidine analog || Takuji || -20°C || || 1:600 - || || || || http://www.antibodies-online.com/antibody/964591/anti-Bromodeoxyuridine+BrDU+antibody/ || <br />
|-<br />
| Casp3 || Abcam || ab13847 || rabbit || || || Lidia || -20C || || || || || || || <br />
|-<br />
| col1A2 || Developmental Studies Hybridoma Bank || SP1.D8 || Mouse || Monoclonal || Sheep || Yuka || -20C || 187ug/ml || 1:200- || 1:200_found in most connective tissue and abundant in bone, cornea, dermis and tendon. || || || || <br />
|-<br />
| col2A1 || Acris || AM00619PU-N || Mouse || || || Yuka || -20C || 0.2mg/ml in /H2O || 1:10 || 1:100-200 || || || || <br />
|-<br />
| Digoxigenin-AP || Roche || 11093274910 || Sheep || Polyclonal || || Common box || 4°C || || || || || || http://www.sigmaaldrich.com/catalog/product/roche/11093274910?lang=de&region=AT&gclid=Cj0KEQjwk-jGBRCbxoPLld_bp-IBEiQAgJaftdEIkv2iCvPxqx7NfQns9MUkipGFBChRXSL1FhsYOxkaApPb8P8HAQ || <br />
|-<br />
| FITC || Jackson ImmunoResearch || 200-002-037 || Mouse || Monoclonal || Fluorescein isothiocyanate || Takuji || -20°C || || 1:400 - || || || || https://www.jacksonimmuno.com/catalog/products/200-002-037 || <br />
|-<br />
| FITC || invitrogen || 71-1900 || Rabbit || Polyclonal || Fluorescein isothiocyanate || Takuji || -20°C || || 1:400 - || || || || https://www.thermofisher.com/antibody/product/FITC-Antibody-Polyclonal/71-1900 || <br />
|-<br />
| GFP || Rockland || 600-401-215 || Rabbit || || || Common box || -20C || || || 1:2000-4000 || || || || <br />
|-<br />
| GFP || MPI || || Goat || || || Yuka (from Jifeng) || -20C || || Yuka (from Jifeng) || 1:2000- || || || || <br />
|-<br />
| GFP || MPI || 106-A20-Mix || Mouse || || || Yuka || -20C || 2.96 mg/ml in PBS || || 1:200, High background || || || || <br />
|-<br />
| GFP || Abcam || ab13970 || Chick || || || Yuka (from Andrea), Lidia || -20C || || Andrea || High background || || || || <br />
|-<br />
| GFP || Torrey Pines || TP401 || Rabbit || || || Yuka || -20C || || 1:200 (Axolotl) || || || || || <br />
|-<br />
| GFP || Invitrogen || A11120 || Mouse || monoclonal || || Common box || -20C || 1 mg/ml in PBS || || || || || || <br />
|-<br />
| Hoxa11 || Tanaka Lab (Kathleen) || || Rabbit || Polyclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 0.95 mg/ml || || FF+2%MEMFA.Dent's || || || || <br />
|-<br />
| Hoxa11 || Tanaka Lab (Kathleen) || || Mouse || Monoclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 4.22 mg/ml || || || || || || <br />
|-<br />
| Hoxa13 || Tanaka Lab (Kathleen) || || Rabbit || Polyclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 0.15 mg/ml || 01:50 || 1:50, FreshFrozen+Unfixed || || || || <br />
|-<br />
| Hoxa9 || Tanaka Lab (Kathleen) || || Rabbit || Polyclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 1.35 mg/ml || || FF+Methanol+ph9AR || || || || <br />
|-<br />
| Laminin || Sigma || L9393 || Rabbit || || || Yuka (from Jifeng) || -20C || || || 1:200, 500_1:25 (paraffin), 1:100 (Slack) || || || || <br />
|-<br />
| MBP || Genetex || GTX76114 || Rat || Monoclonal || Myelin basic protein (Bovine) || Common box || -20C || || 1:200 || 1:200 || || || || <br />
|-<br />
| Mef2c || Sigma || HPA005533 || Rabbit || Polyclonal || Human MEF2C (Myocyte enhancer factor 2C) || Aliquot from Takuji || || || 1:50 (Prayag), 1:300 (Takuji) || 1:5000,10000 (High background on all type of tissue) || || || || <br />
|-<br />
| Meis 1/2/3 || Millipore || 05-779 || Mouse IgG1 || Monoclonal || mouse Meis 1 (aa 60-390) || Lidia || -20C (30% glycerol) || || 1:200 (Takuji?- frozen tissue secitons) || || 1:200 (Lidia?) || || http://www.merckmillipore.com/AT/de/product/Anti-Meis-1%2F2%2F3-Antibody%2C-clone-9.2.7,MM_NF-05-779?ReferrerURL=https%3A%2F%2Fwww.google.at%2F&bd=1 || <br />
|-<br />
| MHC || Developmental Studies Hybridoma Bank || A4.1025 || Mouse || || || Yuka || 4°C || || 1:200 || 1:200 || || || || <br />
|-<br />
| Osterix || abcam || ab22552 || rabbbit || || || Lidia || || || || || || || || <br />
|-<br />
| Pax7 || MPI || || Mouse || || || Yuka || -20C || || 1:100, 200, 400 || || || || || <br />
|-<br />
| PCNA || Santa Cruz || sc-56 AF64 || Alexa Fluor -647 || || || Lidia || || || || 1:500 || || || || <br />
|-<br />
| PDGFRa (16A1) - Biotin || Invitrogen || A15732 || Mouse || Monoclonal || Human CD104a || Yuka (from Josh) || 4°C || 1 mg/ml || || || || || https://www.thermofisher.com/antibody/product/PDGFRA-Antibody-clone-16A1-Monoclonal/A15732 || <br />
|-<br />
| Perilipin || R and D || P1873 || rabbit || || || Lidia || || || || || || || || <br />
|-<br />
| PHH3 || millipore || 06-570 || rabbit || Polyclonal || Linear peptide corresponding to human Histone H3 at Ser10 || Takuji || 4°C || 1 mg/ml || 1:400 - || || || || https://www.merckmillipore.com/DE/de/product/Anti-phospho-Histone-H3-%28Ser10%29-Antibody%2C-Mitosis-Marker,MM_NF-06-570?ReferrerURL=https%3A%2F%2Fwww.emdmillipore.com%2FCA%2Fen%2Fproduct%2FAnti-phospho-Histone-H3-%2528Ser10%2529-Antibody%252C-Mitosis-Marker%2CMM_NF-06-570 || <br />
|-<br />
| PPARgamma || Cell signalling || 81B8 || rabbit || || || Lidia || || || || || || || || <br />
|-<br />
| Prrx1 || Tanaka Lab (Prayag) || || Rabbit || Polyclonal || Axolotl N-terminus (1-101 aa) || Lidia, Yuka_Aliquot from Prayag || -20C || || 1:200 || 1:200 || || || || <br />
|-<br />
| RFP || Rockland || 600-401-379 || Rabbit || Polyclonal || || Common box || -20C || || 1:300-1:1000 for Axolotl || 1:1000 or 1:2000 || || || http://www.rockland-inc.com/Product.aspx?id=34801 || <br />
|-<br />
| Rhodamine || Molecular Probes || A-6397 || Rabbit || Polyclonal || Tetramethylrhodamine || Takuji || -20°C || || 1:400 - || || || || || <br />
|-<br />
| Scleraxis || Santa Cruz || D-14, sc-87425 || Goat || Polyclonal Goat IgG || || Yuka || 4°C || || || || || || || <br />
|-<br />
| Shh (H-160) || Santa Cruz || SC-9024 || Rabbit || Polyclonal || || Yuka || 4°C || || 200 ug/ml || || || || || <br />
|-<br />
| Sox9 || Chemicon || Ab5535 || Rabbit || Polyclonal || Synthetic peptide from human Sox9, aa486-509 (extreme C-Terminus) || Yuka_Aliquot || -20C || || 1:500-1:1000 || 1:500 || || || http://www.merckmillipore.com/DE/en/product/Anti-Sox9-Antibody,MM_NF-AB5535 || <br />
|-<br />
| Sox9 || R&D || AF3075 || Goat || Polyclonal || E. coli-derived recombinant human SOX9 (Met1-Lys151) || Lidia, Yuka || -20C || || 1:100-1:200 || 1:100 || || || https://www.rndsystems.com/products/human-sox9-antibody_af3075 || <br />
|-<br />
| Zo-1 || Invitrogen || 33-9100 || Mouse || || || Yuka (from Akira) || -20C || || || || || || || <br />
|-<br />
| GADD45B || SIGMA || SAB2108614-100 || Rabbit || Polyclonal || QIHFT LIQSF CCDND INIVR VSGMQ RLAQL LGEPA ETQGT TEARD LHCLL || Marco || -20C || 0.5-1 mg/ml || || || || || || <br />
|-<br />
| GADD45G || SIGMA || HPA023606 || Rabbit || Polyclonal || MTLEE VRGQD TVPES TARMQ GAGKA LHELL LSAQR QGCLT AGVYE SAKVL NVDPD NVTFC VLAAG EEDEG DIALQ IHFTL IQAFC CENDI DIVRV GDVQR LAAIV G || Marco || -20C || 0.05mg/ml || || || || || || <br />
|-<br />
| TDG || SIGMA || HPA052263 || Rabbit || Polyclonal || WKCLF MSGLS EVQLN HMDDH TLPGK YGIGF TNMVE RTTPG SKDLS SKEFR EGGRI LVQKL QKYQP RIAVF NGKCI YEIFS KEVFG VKVKN LEFG || || -20C || 0.1mg/ml || || || || || || <br />
|-<br />
| Tet3 || Diagenode || C15410311 || Rabbit || Polyclonal || || Marco || -20C || 1 ug/uL || || || || || || <br />
|-<br />
| Tet2 || Diagenode || C15410255 || Rabbit || Polyclonal || || Marco || -20C || 1 ug/uL || || || || || || <br />
|-<br />
| 5-mC || Diagenode || C15200081-100 || Mouse || Monoclonal || || Marco || -20C || 1.36 ug/ uL || || || || || || <br />
|-<br />
| 5-hmC || Diagenode || C15410205-20 || Rabbit || Polyclonal || || Marco || -20C || 3.5 ug/uL || || || || || || <br />
|-<br />
| IgG (Rabbit) || Diagenode || C15410206 || Rabbit || Polyclonal || || Marco || -20C || 1 ug/uL || || || || || || <br />
|}<br />
<br />
== Protocols ==<br />
<ul><br />
=== Cell culture ===<br />
<li>[[Adhesive_cell_culture|Adhesive cell culture]]</li><br />
<li>[[Baculovirus_titration|Baculovirus titration]]</li><br />
<li>[[Cardiomyocyte_Preparation|Cardiomyocyte preparation]]</li><br />
<li>[[Electroporation_of_nec|Electroporation of neural epithelia cells (axolotl spinal cord)]]</li><br />
<li>[[Myoblasts_electroporation|Myoblasts electroporation]]</li><br />
<li>[[Neurosphere_culture|Neurosphere culture]]</li><br />
<li>[[Thawing_293FT_cells|Thawing 293FT cells]]</li><br />
<li>[[G418_medium_preparation|G418 medium preparation]]</li><br />
<li>[[Fibronectin_Preparation|Fibronectin preparation]]</li><br />
<li>[[Gelatin Preparation|Gelatin preparation]]</li><br />
<li>[[Insulin_Preparation|Insulin preparation]]</li> <br />
<li>[[Insulin_Preparation|Insulin preparation]]</li> <br />
<li>[[Gelatin Embedding|Gelatin Embedding]]</li> <br />
<br />
===== A1 cells =====<br />
<br />
<li>[[Thawing_frozen_A1_cells|Thawing frozen A1 cells]]</li><br />
<li>[[Freezing_of_A1_cells|Freezing of A1 cells]]</li><br />
<li>[[Passaging_A1_cell|A1 cell passaging]]</li><br />
<li>[[A1_cell_passaging_myotube|A1 cell passaging for myotube preparation]]</li><br />
<li>[[Modified_myotube_prep|Modified myotube prep]]</li><br />
<li>[[A1_cell_transfection_with_FuGene|A1 cell transfection with FuGene]]</li><br />
<li>[[Electroporation_of_A1_cells|Electroporation of A1 cells]]</li><br />
<li>[[BrdU_labelling_of_A1_cells_in_96-well_plate|BrdU labelling of A1 cells in 96-well plate]]</li><br />
<li>[[BrdU_and_myosin_staining|BrdU and myosin staining]]</li><br />
<li>[[Preparation_of_Low_Serum_for_A1_cells|Preparation of Low Serum for A1 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_A1_cells|Preparation of High Serum for A1 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_A1_cells|Preparation of Freezing media for A1 cells]]</li><br />
<br />
===== Blastema cells =====<br />
<li>[[Blastema_cells|Blastema cells]]</li><br />
<li>[[Electroporation_of_blastema_cells|Electroporation of blastema cells]]</li><br />
<br />
===== C2C12 cells =====<br />
<br />
<li>[[Thawing_frozen_C2C12_cells|Thawing frozen C2C12 cells]]</li><br />
<li>[[Freezing_of_C2C12_cells|Freezing of C2C12 cells]]</li><br />
<li>[[Passaging_C2C12_cells|C2C12 cell passaging]]</li><br />
<li>[[Myotube_preparation_of_C2C12_cells|Myotube preparation of C2C12 cells]]</li><br />
<li>[[C2C12_cell_transfection_with_FuGene|C2C12 cell transfection with FuGene]]</li><br />
<li>[[Cloning_of_C2C12_cells|Cloning of C2C12 cells]]</li><br />
<li>[[Electroporation_of_C2C12_cells|Electroporation of C2C12 cells]]</li><br />
<li>[[Preparation_of_Low_Serum_for_C2C12_cells|Preparation of Low Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_C2C12_cells|Preparation of High Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_C2C12_cells|Preparation of Freezing media for C2C12 cells]]</li><br />
<br />
===== Embryonic stem cells =====<br />
<li>[[mESC_culture|mESC culture/Cyst formation]]</li><br />
<br />
===== Hybridoma cells =====<br />
<li>[[Thawing_(XB10)_hybridoma_cells|Thawing (XB10) hybridoma cells]]</li><br />
<li>[[Hybridoma_cells_in_SFX_media|Hybridoma cells in SFX media]]</li><br />
<li>[[Hybridoma_cells_clones|Hybridoma cell clones]]</li><br />
<br />
=== Histology ===<br />
<li>[[Alcian_Blue_Staining|Alcian Blue Staining]]</li><br />
<li>[[Double_Immunostaining_via_antigen_retrieval|Double Immunostaining via antigen retrieval (Sherry)]]</li><br />
<li>[[TSA_Staining_on_axolotl-tissue|Tyramide signal amplification (TSA) staining on axolotl tissue]]</li><br />
<br />
===== In situ Hybridization =====<br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Anja)]]</li><br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Akira)]]</li><br />
<li>[[Whole_mount_ISH_on_axolotl_tissue|Whole mount ISH on axolotl tissue]]</li><br />
<br />
=== Antibodies ===<br />
<br />
===== Monoclonal Antibodies =====<br />
<li>[[Purifying IgG1 from Hybridoma Supernatant]]</li><br />
<li>[[Labelling myosin antibody]]</li><br />
<li>[[Labelling Pax6 with Dig-NHS]]</li><br />
<li>[[Anti-BrDU Antibody labeling]]</li><br />
<br />
===== Polyclonal Antibodies =====<br />
<li>[[Address for making polyclonal antibodies]]</li><br />
<li>[[Letter to Froppier (French)]]</li><br />
<li>[[labeled AB staining]]</li><br />
<li>[[Antibody staining]]</li><br />
<li>[[Antibody purification]]</li><br />
<li>[[Antibody purification Sox2]]</li><br />
<br />
===== ELISA =====<br />
<li>[[ELISA]]</li><br />
<br />
=== Molecular biology ===<br />
<li>[[Metamorphosis_protocol|Metamorphosis protocol]]</li><br />
<li>[[Digestion_DNA|Plasmid digestion]]</li><br />
<li>[[RNA_Extraction_from_axolotl_tissue|RNA Extraction (axolotl tissue)]]</li><br />
<li>[[RNA_formaldehyde_gels|RNA formaldehyde gels]]</li><br />
<li>[[Amputation_andRNA_Isolation|Amputation and RNA Isolation]]</li><br />
<li>[[Linker Insertion|Linker Insertion]]</li><br />
<li>[[gRNA Production|gRNA Production]]</li><br />
<li>[[Cocktail Protocol for Pax7|Cocktail Protocol for Pax7]]</li><br />
<br />
== Genetics ==<br />
=== Transgenic lines ===<br />
<ul><br />
<li>[[Transgenic_axolotl_lines|Axolotl (A.mexicanum)]]</li><br />
<li>[[Transgenic_frog_lines|Frog (X.laevis)]]</li><br />
</ul><br />
<br />
=== Markers ===<br />
{| class="wikitable sortable"<br />
!style="width: 150px" | Marker<br />
!style="width: 250px" | Full name<br />
!style="width: 250px" | Target<br />
!style="width: 450px" | Remarks<br />
!style="width: 200px" | Link<br />
|-<br />
|DAPI/Hoechst<br />
|4',6-diamidino-2-phenylindole<br />
|Nuclei<br />
|Incorporates into the DNA<br />
|[http://en.wikipedia.org/wiki/DAPI DAPI], [http://en.wikipedia.org/wiki/Hoechst_stain Hoechst]<br />
|-<br />
|PCNA<br />
|Proliferating-Cell-Nuclear-Antigen<br />
|Dividing cells<br />
|<br />
|[http://en.wikipedia.org/wiki/PCNA PCNA]<br />
|-<br />
|SOX2<br />
|SRY (sex determining region Y)-box 2<br />
|Preferentially neuronal lineage<br />
|Progenitor marker for both spinal cord and brain<br />
|[http://en.wikipedia.org/wiki/Sox2 SOX2]<br />
|-<br />
|PAX6<br />
|Paired box protein Pax-6<br />
|Brain/spinal cord<br />
|aka aniridia type II protein (AN2) or oculorhombin<br />
|[http://en.wikipedia.org/wiki/Pax6 PAX6]<br />
|-<br />
|PAX7<br />
|Paired box protein Pax-7<br />
|Satellite cells/spinal cord/brain<br />
|<br />
|[http://en.wikipedia.org/wiki/PAX7 PAX7]<br />
|-<br />
|MHC<br />
|Myosin heavy chain<br />
|Muscle fibers<br />
|<br />
|[http://en.wikipedia.org/wiki/Myosin_heavy_chain MHC]<br />
|-<br />
|MYF5<br />
|Myogenic factor 5<br />
|Myoblasts<br />
|Regulates muscle differentiation, labels cycling myoblasts<br />
|[http://en.wikipedia.org/wiki/Myf5 MYF5]<br />
|-<br />
|TUBB3<br />
|ßIII-tubulin<br />
|Axons<br />
|<br />
|[http://en.wikipedia.org/wiki/TUBB3 TUBB3]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Schwann cells<br />
|<br />
|-<br />
|PRRX1<br />
|Paired mesoderm homeobox protein 1<br />
|Connective tissue progenitors in Limb bud and Limb Blastema<br />
|<br />
|[http://en.wikipedia.org/wiki/PRRX1 PRRX1]<br />
|-<br />
|SMA<br />
|Smooth muscle actin<br />
|Blood vessels/endothelial cells<br />
|<br />
|<br />
|-<br />
|GFAP<br />
|Glial fibrillary acidic protein<br />
|Glia<br />
|<br />
|[http://en.wikipedia.org/wiki/Glial_fibrillary_acidic_protein GFAP]<br />
|-<br />
|NeuN<br />
|Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3<br />
|Differentiated nerve<br />
|<br />
|[http://en.wikipedia.org/wiki/NeuN NeuN]<br />
|-<br />
|LEU7/HNK1/B3GAT1/CD57<br />
|Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1<br />
|Schwann cells<br />
|<br />
|[http://en.wikipedia.org/wiki/B3GAT1 B3GAT1]<br />
|-<br />
|XBRA<br />
|Xbrachyury<br />
|Mesoderm during gastrula stage<br />
|<br />
|[http://en.wikipedia.org/wiki/Brachyury Brachyury]<br />
|-<br />
|MSX1<br />
|Muscle segment homeobox, Msh homeobox 1<br />
|Undifferentiated cells<br />
|Transcriptional repressor during embryogenesis<br />
|[http://en.wikipedia.org/wiki/MSX1 MSX1]<br />
|-<br />
|Trypan blue<br />
|<br />
|Dead tissues or cells<br />
|Vital stain<br />
|[http://en.wikipedia.org/wiki/Trypan_blue Trypan blue]<br />
|-<br />
|MEF2C<br />
|Myocyte-specific enhancer factor 2C<br />
|Differentiated muscle cells<br />
|aka MADS box transcription enhancer factor 2, polypeptide C<br />
|[http://en.wikipedia.org/wiki/Mef2c MEF2C]<br />
|-<br />
|PH3<br />
|Phospho-Histone H3<br />
|Mitotic cells<br />
|<br />
|<br />
|-<br />
|BrdU<br />
|Bromodeoxyuridine<br />
|Cells in S-phase<br />
|<br />
|[http://en.wikipedia.org/wiki/Brdu BrdU]<br />
|-<br />
|CASP3<br />
|Caspase 3<br />
|Dying cells<br />
|<br />
|[http://en.wikipedia.org/wiki/CASP3 CASP3]<br />
|-<br />
|GDF5<br />
|Growth/differentiation factor 5<br />
|Joints<br />
|Joint development marker<br />
|[http://en.wikipedia.org/wiki/GDF5 GDF5]<br />
|-<br />
|Survivin<br />
|<br />
|Cleavage furrow<br />
|aka baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5<br />
|[http://en.wikipedia.org/wiki/Survivin Survivin]<br />
|-<br />
|MAP2<br />
|Microtubule-associated protein 2<br />
|Neurons<br />
|<br />
|[http://en.wikipedia.org/wiki/MAP2 MAP2]<br />
|-<br />
|SOX9<br />
|SRY (sex determining region Y)-box 2<br />
|Cartillage progenitors<br />
|<br />
|[http://en.wikipedia.org/wiki/Sox9 SOX9]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Myelin sheath<br />
|<br />
|[http://en.wikipedia.org/wiki/Myelin_basic_protein MBP]<br />
|}<br />
<br />
== Digital data ==<br />
Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on. <br />
Select a category from the list below in order to view or download the data.<br />
<br />
=== Literature ===<br />
<ul><br />
<li>[[Literature#Theses|Theses]]</li><br />
<li>[[Literature#Publications|Publications]]</li><br />
</ul><br />
<br />
=== Databases ===<br />
<ul><br />
<li>[https://axolotl-omics.org Axolotl transcriptome] website contains the Axolotl transcriptome assemblies</li><br />
<li>[https://genome.axolotl-omics.org Axolotl genome] website contains the Axolotl genome assemblies</li><br />
<li>[http://newtomics.mpi-bn.mpg.de/ Newt transcriptome] website contains the Newt transcriptome assembly</li><br />
<li>[http://sandberg.cmb.ki.se/redspottednewt/ Red spotted newt transcriptome] website contains the red spotted newt transcriptome assembly</li><br />
<li>[https://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi Axolotl EST] database contains several ESTs and also some additional information, e.g. the position of the well with the corresponding insert in the Blastema (BL) or Neural tube (NT) library.</li><br />
<li>[http://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi?dispatch=contig_search.cgi Short insert cDNA library]</li><br />
<li>[https://axologl.axolotl-omics.org Axologle] database contains expression profiling data of Axolotl limb regeneration</li><br />
<li>[https://axologl.axolotl-omics.org/blast/blast.html Axolotl BLAST] database contains several different BLAST-able Axolotl transciptome assemblies</li><br />
</ul><br />
<br />
=== Datasets ===<br />
<ul><br />
<li>[[Datasets#Sequences|Sequences]]<br />
<ul><br />
<li>[[Datasets#Illumina|Illumina]]</li><br />
<li>[[Datasets#Sanger|Sanger]]</li><br />
<li>[[Datasets#Roche.2F454|Roche/454]]</li><br />
</ul><br />
</li><br />
<li>[[Datasets#Microarrays|Microarrays]]<br />
</ul><br />
<br />
=== Interesting links ===<br />
<ul><br />
<li>[http://www.nytimes.com/video/2013/02/27/science/100000002087758/finding-the-visible-in-the-invisible.html Visible in the invisible]: a movie introducing a new image processing ana analysis technique developed at the MIT.</li><br />
</ul></div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Overview&diff=914Overview2020-05-19T11:21:23Z<p>Sergej.nowoshilow: /* List of primary antibodies */</p>
<hr />
<div>== Introduction ==<br />
On the pages listed below you can find most common protocols used in the lab as well as a list of lab duties along with the names of the responsible persons. Please, contact the responsible persons if you have any question, suggestions or troubles.<br />
<br />
== Axolotl ==<br />
<br />
=== Maintainance ===<br />
In this section you will find some general information on how to [[Axolotl#Maintaining_the_axolotls|maintain]] the animals.<br />
<br />
=== Mating ===<br />
In this section you will find the information on how to [[Axolotl#Mating_the_axolotls|mate]] axolotls.<br />
<br />
=== Feeding ===<br />
This section will give you an overview of how to [[Axolotl#Feeding|feed]] the axolotls.<br />
<br />
=== Axolotl in Vitro Fertilization ===<br />
This section will give you an overview of how to [[Axolotl#In_vitro_fertilization|In vitro fertilize]] the axolotls.<br />
<br />
=== Interesting observations ===<br />
In the following section you can find some [[Axolotl#Interesting_observations|interesting observations]] concerning the axolotl, its behavior, caveats of the transgenics and many more.<br />
<br />
=== Genotyping (galaxy) ===<br />
This section section will give you an overview of how to [[Axolotl#Genotyping_(galaxy)|genotype]] the axolotls.<br />
<br />
== Lab duties ==<br />
<ul><br />
=== Chemicals ===<br />
<li>[[Antibiotics|Antibiotics]]</li><br />
<li>[[Benzocaine|Benzocaine]]</li><br />
<li>[[Fibronectin|Fibronectin]]</li><br />
<li>[[Gelatine|Gelatine]]</li><br />
<li>[[Liquid_nitrogen|Liquid nitrogen]]</li><br />
<li>[[Primary_antibodies|Primary antibodies]]</li><br />
<li>[[Secondary_antibodies|Secondary antibodies]]</li><br />
<li>[[Serum_heat_inactivation|Serum heat inactivation]]</li><br />
<br />
=== Hardware ===<br />
<li>[[Autoclave|Autoclave]]</li><br />
<li>[[Cryostat|Cryostat]]</li><br />
<li>[[Dissecting_microscopes|Dissecting microscopes]]</li><br />
<li>[[Leica_confocal_microscope|Leica confocal microscope]]</li><br />
<li>[[Vacuum_pumps|Vacuum pumps]]</li><br />
<li>[[Waterbath|Waterbath]]</li><br />
<li>[[RAMP Test|RAMP Test]]</li><br />
<br />
=== General ===<br />
<li>[[Ordering_common_stuff|Ordering common stuff]]</li><br />
<li>[[Heat_shock_competent_cells|Heat-shock competent cells]]</li><br />
</ul><br />
<br />
== Antibodies ==<br />
=== List of primary antibodies ===<br />
{| class="wikitable sortable"<br />
! Name !! Supplier !! Cat. No !! Host !! Mono/Poly !! Antigen !! class="unsortable" | Contact person !! class="unsortable" | Storage temp !! class="unsortable" | Stock concentration !! class="unsortable" | Conditions for axolotl !! class="unsortable" | Conditions for Xenopus !! class="unsortable" | Conditions for mouse !! class="unsortable" | Conditions for cell culture !! class="unsortable" | Reference<br />
|-<br />
| 12/101 || Developmental Studies Hybridoma Bank || || Mouse || || || Yuka || 4°C || 46 ug/ml Ig || 1:200 || 1:200 || || || || <br />
|-<br />
| 15.3B9 (NOT1) || Developmental Studies Hybridoma Bank || 15.3B9 (NOT1) || Mouse || Monoclonal || notochord marker (Chicken) || Yuka || 4°C || || 21 ug/ml IG || || || || http://dshb.biology.uiowa.edu/notochord-marker || <br />
|-<br />
| 20B4 || Developmental Studies Hybridoma Bank || 20B4 || Mouse || Monoclonal || neural crest cell (Chicken) || Yuka || 4°C || || || || || || http://dshb.biology.uiowa.edu/neural-crest-cells || <br />
|-<br />
| 5D3 || Developmental Studies Hybridoma Bank || 5D3 || Mouse || Monoclonal || Cadherin E (Xenopus) || Yuka || 4°C || || 37 ug/ml IG || || || || http://dshb.biology.uiowa.edu/5D3 || <br />
|-<br />
| 8C8 || Developmental Studies Hybridoma Bank || 8C8 || Mouse || Monoclonal || integrin beta-1/ CD29 (Xenopus) || Yuka || 4°C || || || || || || http://dshb.biology.uiowa.edu/integrin-beta-1_7 || <br />
|-<br />
| Aggrecan || millipore || AB1031 || Rabbit || || || Lidia || || || || Not work, Dim || '1:500 (Lidia) || || || <br />
|-<br />
| Alpha tubulin (DM1a) || MPI || || Mouse || Monoclonal || Chicken alpha tubulin || Takuji || -20°C || || || || || 1:1000 - || || <br />
|-<br />
| b-catenin || Gift from Thomas Kurth || P14L || Rabbit || || Xenopus || Yuka_Aliquot from Tomas Kurth || || || || || || || Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. || <br />
|-<br />
| Bra || || AF2085 || || || || Yuka || -20C || || 0.2ug/ml in PBS || || || || || <br />
|-<br />
| BrdU (Bu20a) || MPI || || Mouse || Monoclonal || BrdU - thymidine analog || Takuji || -20°C || || 1:400 - || || || || || <br />
|-<br />
| BrdU || Antibodies-online || ABIN964591 || Rabbit || Polyclonal || BrdU - thymidine analog || Takuji || -20°C || || 1:600 - || || || || http://www.antibodies-online.com/antibody/964591/anti-Bromodeoxyuridine+BrDU+antibody/ || <br />
|-<br />
| Casp3 || Abcam || ab13847 || rabbit || || || Lidia || -20C || || || || || || || <br />
|-<br />
| col1A2 || Developmental Studies Hybridoma Bank || SP1.D8 || Mouse || Monoclonal || Sheep || Yuka || -20C || 187ug/ml || 1:200- || 1:200_found in most connective tissue and abundant in bone, cornea, dermis and tendon. || || || || <br />
|-<br />
| col2A1 || Acris || AM00619PU-N || Mouse || || || Yuka || -20C || 0.2mg/ml in /H2O || 1:10 || 1:100-200 || || || || <br />
|-<br />
| Digoxigenin-AP || Roche || 11093274910 || Sheep || Polyclonal || || Common box || 4°C || || || || || || http://www.sigmaaldrich.com/catalog/product/roche/11093274910?lang=de&region=AT&gclid=Cj0KEQjwk-jGBRCbxoPLld_bp-IBEiQAgJaftdEIkv2iCvPxqx7NfQns9MUkipGFBChRXSL1FhsYOxkaApPb8P8HAQ || <br />
|-<br />
| FITC || Jackson ImmunoResearch || 200-002-037 || Mouse || Monoclonal || Fluorescein isothiocyanate || Takuji || -20°C || || 1:400 - || || || || https://www.jacksonimmuno.com/catalog/products/200-002-037 || <br />
|-<br />
| FITC || invitrogen || 71-1900 || Rabbit || Polyclonal || Fluorescein isothiocyanate || Takuji || -20°C || || 1:400 - || || || || https://www.thermofisher.com/antibody/product/FITC-Antibody-Polyclonal/71-1900 || <br />
|-<br />
| GFP || Rockland || 600-401-215 || Rabbit || || || Common box || -20C || || || 1:2000-4000 || || || || <br />
|-<br />
| GFP || MPI || || Goat || || || Yuka (from Jifeng) || -20C || || Yuka (from Jifeng) || 1:2000- || || || || <br />
|-<br />
| GFP || MPI || 106-A20-Mix || Mouse || || || Yuka || -20C || 2.96 mg/ml in PBS || || 1:200, High background || || || || <br />
|-<br />
| GFP || Abcam || ab13970 || Chick || || || Yuka (from Andrea), Lidia || -20C || || Andrea || High background || || || || <br />
|-<br />
| GFP || Torrey Pines || TP401 || Rabbit || || || Yuka || -20C || || 1:200 (Axolotl) || || || || || <br />
|-<br />
| GFP || Invitrogen || A11120 || Mouse || monoclonal || || Common box || -20C || 1 mg/ml in PBS || || || || || || <br />
|-<br />
| Hoxa11 || Tanaka Lab (Kathleen) || || Rabbit || Polyclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 0.95 mg/ml || || FF+2%MEMFA.Dent's || || || || <br />
|-<br />
| Hoxa11 || Tanaka Lab (Kathleen) || || Mouse || Monoclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 4.22 mg/ml || || || || || || <br />
|-<br />
| Hoxa13 || Tanaka Lab (Kathleen) || || Rabbit || Polyclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 0.15 mg/ml || 01:50 || 1:50, FreshFrozen+Unfixed || || || || <br />
|-<br />
| Hoxa9 || Tanaka Lab (Kathleen) || || Rabbit || Polyclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 1.35 mg/ml || || FF+Methanol+ph9AR || || || || <br />
|-<br />
| Laminin || Sigma || L9393 || Rabbit || || || Yuka (from Jifeng) || -20C || || || 1:200, 500_1:25 (paraffin), 1:100 (Slack) || || || || <br />
|-<br />
| MBP || Genetex || GTX76114 || Rat || Monoclonal || Myelin basic protein (Bovine) || Common box || -20C || || 1:200 || 1:200 || || || || <br />
|-<br />
| Mef2c || Sigma || HPA005533 || Rabbit || Polyclonal || Human MEF2C (Myocyte enhancer factor 2C) || Aliquot from Takuji || || || 1:50 (Prayag), 1:300 (Takuji) || 1:5000,10000 (High background on all type of tissue) || || || || <br />
|-<br />
| Meis 1/2/3 || Millipore || 05-779 || Mouse IgG1 || Monoclonal || mouse Meis 1 (aa 60-390) || Lidia || -20C (30% glycerol) || || 1:200 (Takuji?- frozen tissue secitons) || || 1:200 (Lidia?) || || http://www.merckmillipore.com/AT/de/product/Anti-Meis-1%2F2%2F3-Antibody%2C-clone-9.2.7,MM_NF-05-779?ReferrerURL=https%3A%2F%2Fwww.google.at%2F&bd=1 || <br />
|-<br />
| MHC || Developmental Studies Hybridoma Bank || A4.1025 || Mouse || || || Yuka || 4°C || || 1:200 || 1:200 || || || || <br />
|-<br />
| Osterix || abcam || ab22552 || rabbbit || || || Lidia || || || || || || || || <br />
|-<br />
| Pax7 || MPI || || Mouse || || || Yuka || -20C || || 1:100, 200, 400 || || || || || <br />
|-<br />
| PCNA || Santa Cruz || sc-56 AF64 || Alexa Fluor -647 || || || Lidia || || || || 1:500 || || || || <br />
|-<br />
| PDGFRa (16A1) - Biotin || Invitrogen || A15732 || Mouse || Monoclonal || Human CD104a || Yuka (from Josh) || 4°C || 1 mg/ml || || || || || https://www.thermofisher.com/antibody/product/PDGFRA-Antibody-clone-16A1-Monoclonal/A15732 || <br />
|-<br />
| Perilipin || R and D || P1873 || rabbit || || || Lidia || || || || || || || || <br />
|-<br />
| PHH3 || millipore || 06-570 || rabbit || Polyclonal || Linear peptide corresponding to human Histone H3 at Ser10 || Takuji || 4°C || 1 mg/ml || 1:400 - || || || || https://www.merckmillipore.com/DE/de/product/Anti-phospho-Histone-H3-%28Ser10%29-Antibody%2C-Mitosis-Marker,MM_NF-06-570?ReferrerURL=https%3A%2F%2Fwww.emdmillipore.com%2FCA%2Fen%2Fproduct%2FAnti-phospho-Histone-H3-%2528Ser10%2529-Antibody%252C-Mitosis-Marker%2CMM_NF-06-570 || <br />
|-<br />
| PPARgamma || Cell signalling || 81B8 || rabbit || || || Lidia || || || || || || || || <br />
|-<br />
| Prrx1 || Tanaka Lab (Prayag) || || Rabbit || Polyclonal || Axolotl N-terminus (1-101 aa) || Lidia, Yuka_Aliquot from Prayag || -20C || || 1:200 || 1:200 || || || || <br />
|-<br />
| RFP || Rockland || 600-401-379 || Rabbit || Polyclonal || || Common box || -20C || || 1:300-1:1000 for Axolotl || 1:1000 or 1:2000 || || || http://www.rockland-inc.com/Product.aspx?id=34801 || <br />
|-<br />
| Rhodamine || Molecular Probes || A-6397 || Rabbit || Polyclonal || Tetramethylrhodamine || Takuji || -20°C || || 1:400 - || || || || || <br />
|-<br />
| Scleraxis || Santa Cruz || D-14, sc-87425 || Goat || Polyclonal Goat IgG || || Yuka || 4°C || || || || || || || <br />
|-<br />
| Shh (H-160) || Santa Cruz || SC-9024 || Rabbit || Polyclonal || || Yuka || 4°C || || 200 ug/ml || || || || || <br />
|-<br />
| Sox9 || Chemicon || Ab5535 || Rabbit || Polyclonal || Synthetic peptide from human Sox9, aa486-509 (extreme C-Terminus) || Yuka_Aliquot || -20C || || 1:500-1:1000 || 1:500 || || || http://www.merckmillipore.com/DE/en/product/Anti-Sox9-Antibody,MM_NF-AB5535 || <br />
|-<br />
| Sox9 || R&D || AF3075 || Goat || Polyclonal || E. coli-derived recombinant human SOX9 (Met1-Lys151) || Lidia, Yuka || -20C || || 1:100-1:200 || 1:100 || || || https://www.rndsystems.com/products/human-sox9-antibody_af3075 || <br />
|-<br />
| Zo-1 || Invitrogen || 33-9100 || Mouse || || || Yuka (from Akira) || -20C || || || || || || || <br />
|-<br />
| GADD45B || SIGMA || SAB2108614-100 || Rabbit || Polyclonal || QIHFT LIQSF CCDND INIVR VSGMQ RLAQL LGEPA ETQGT TEARD LHCLL || Marco || -20C || 0.5-1 mg/ml || || || || || || <br />
|-<br />
| GADD45G || SIGMA || HPA023606 || Rabbit || Polyclonal || MTLEE VRGQD TVPES TARMQ GAGKA LHELL LSAQR QGCLT AGVYE SAKVL NVDPD NVTFC VLAAG EEDEG DIALQ IHFTL IQAFC CENDI DIVRV GDVQR LAAIV G || Marco || -20C || 0.05mg/ml || || || || || || <br />
|-<br />
| TDG || SIGMA || HPA052263 || Rabbit || Polyclonal || WKCLF MSGLS EVQLN HMDDH TLPGK YGIGF TNMVE RTTPG SKDLS SKEFR EGGRI LVQKL QKYQP RIAVF NGKCI YEIFS KEVFG VKVKN LEFG || || -20C || 0.1mg/ml || || || || || || <br />
|-<br />
| Tet3 || Diagenode || C15410311 || Rabbit || Polyclonal || || Marco || -20C || 1 ug/uL || || || || || || <br />
|-<br />
| Tet2 || Diagenode || C15410255 || Rabbit || Polyclonal || || Marco || -20C || 1 ug/uL || || || || || || <br />
|-<br />
| 5-mC || Diagenode || C15200081-100 || Mouse || Monoclonal || || Marco || -20C || 1.36 ug/ uL || || || || || || <br />
|-<br />
| 5-hmC || Diagenode || C15410205-20 || Rabbit || Polyclonal || || Marco || -20C || 3.5 ug/uL || || || || || || <br />
|-<br />
| IgG (Rabbit) || Diagenode || C15410206 || Rabbit || Polyclonal || || Marco || -20C || 1 ug/uL || || || || || || <br />
|}<br />
<br />
== Protocols ==<br />
<ul><br />
=== Cell culture ===<br />
<li>[[Adhesive_cell_culture|Adhesive cell culture]]</li><br />
<li>[[Baculovirus_titration|Baculovirus titration]]</li><br />
<li>[[Cardiomyocyte_Preparation|Cardiomyocyte preparation]]</li><br />
<li>[[Electroporation_of_nec|Electroporation of neural epithelia cells (axolotl spinal cord)]]</li><br />
<li>[[Myoblasts_electroporation|Myoblasts electroporation]]</li><br />
<li>[[Neurosphere_culture|Neurosphere culture]]</li><br />
<li>[[Thawing_293FT_cells|Thawing 293FT cells]]</li><br />
<li>[[G418_medium_preparation|G418 medium preparation]]</li><br />
<li>[[Fibronectin_Preparation|Fibronectin preparation]]</li><br />
<li>[[Gelatin Preparation|Gelatin preparation]]</li><br />
<li>[[Insulin_Preparation|Insulin preparation]]</li> <br />
<li>[[Insulin_Preparation|Insulin preparation]]</li> <br />
<li>[[Gelatin Embedding|Gelatin Embedding]]</li> <br />
<br />
===== A1 cells =====<br />
<br />
<li>[[Thawing_frozen_A1_cells|Thawing frozen A1 cells]]</li><br />
<li>[[Freezing_of_A1_cells|Freezing of A1 cells]]</li><br />
<li>[[Passaging_A1_cell|A1 cell passaging]]</li><br />
<li>[[A1_cell_passaging_myotube|A1 cell passaging for myotube preparation]]</li><br />
<li>[[Modified_myotube_prep|Modified myotube prep]]</li><br />
<li>[[A1_cell_transfection_with_FuGene|A1 cell transfection with FuGene]]</li><br />
<li>[[Electroporation_of_A1_cells|Electroporation of A1 cells]]</li><br />
<li>[[BrdU_labelling_of_A1_cells_in_96-well_plate|BrdU labelling of A1 cells in 96-well plate]]</li><br />
<li>[[BrdU_and_myosin_staining|BrdU and myosin staining]]</li><br />
<li>[[Preparation_of_Low_Serum_for_A1_cells|Preparation of Low Serum for A1 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_A1_cells|Preparation of High Serum for A1 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_A1_cells|Preparation of Freezing media for A1 cells]]</li><br />
<br />
===== Blastema cells =====<br />
<li>[[Blastema_cells|Blastema cells]]</li><br />
<li>[[Electroporation_of_blastema_cells|Electroporation of blastema cells]]</li><br />
<br />
===== C2C12 cells =====<br />
<br />
<li>[[Thawing_frozen_C2C12_cells|Thawing frozen C2C12 cells]]</li><br />
<li>[[Freezing_of_C2C12_cells|Freezing of C2C12 cells]]</li><br />
<li>[[Passaging_C2C12_cells|C2C12 cell passaging]]</li><br />
<li>[[Myotube_preparation_of_C2C12_cells|Myotube preparation of C2C12 cells]]</li><br />
<li>[[C2C12_cell_transfection_with_FuGene|C2C12 cell transfection with FuGene]]</li><br />
<li>[[Cloning_of_C2C12_cells|Cloning of C2C12 cells]]</li><br />
<li>[[Electroporation_of_C2C12_cells|Electroporation of C2C12 cells]]</li><br />
<li>[[Preparation_of_Low_Serum_for_C2C12_cells|Preparation of Low Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_C2C12_cells|Preparation of High Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_C2C12_cells|Preparation of Freezing media for C2C12 cells]]</li><br />
<br />
===== Embryonic stem cells =====<br />
<li>[[mESC_culture|mESC culture/Cyst formation]]</li><br />
<br />
===== Hybridoma cells =====<br />
<li>[[Thawing_(XB10)_hybridoma_cells|Thawing (XB10) hybridoma cells]]</li><br />
<li>[[Hybridoma_cells_in_SFX_media|Hybridoma cells in SFX media]]</li><br />
<li>[[Hybridoma_cells_clones|Hybridoma cell clones]]</li><br />
<br />
=== Histology ===<br />
<li>[[Alcian_Blue_Staining|Alcian Blue Staining]]</li><br />
<li>[[Double_Immunostaining_via_antigen_retrieval|Double Immunostaining via antigen retrieval (Sherry)]]</li><br />
<li>[[TSA_Staining_on_axolotl-tissue|Tyramide signal amplification (TSA) staining on axolotl tissue]]</li><br />
<br />
===== In situ Hybridization =====<br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Anja)]]</li><br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Akira)]]</li><br />
<li>[[Whole_mount_ISH_on_axolotl_tissue|Whole mount ISH on axolotl tissue]]</li><br />
<br />
=== Antibodies ===<br />
<br />
===== Monoclonal Antibodies =====<br />
<li>[[Purifying IgG1 from Hybridoma Supernatant]]</li><br />
<li>[[Labelling myosin antibody]]</li><br />
<li>[[Labelling Pax6 with Dig-NHS]]</li><br />
<li>[[Anti-BrDU Antibody labeling]]</li><br />
<br />
===== Polyclonal Antibodies =====<br />
<li>[[Address for making polyclonal antibodies]]</li><br />
<li>[[Letter to Froppier (French)]]</li><br />
<li>[[labeled AB staining]]</li><br />
<li>[[Antibody staining]]</li><br />
<li>[[Antibody purification]]</li><br />
<li>[[Antibody purification Sox2]]</li><br />
<br />
===== ELISA =====<br />
<li>[[ELISA]]</li><br />
<br />
=== Molecular biology ===<br />
<li>[[Metamorphosis_protocol|Metamorphosis protocol]]</li><br />
<li>[[Digestion_DNA|Plasmid digestion]]</li><br />
<li>[[RNA_Extraction_from_axolotl_tissue|RNA Extraction (axolotl tissue)]]</li><br />
<li>[[RNA_formaldehyde_gels|RNA formaldehyde gels]]</li><br />
<li>[[Amputation_andRNA_Isolation|Amputation and RNA Isolation]]</li><br />
<li>[[Linker Insertion|Linker Insertion]]</li><br />
<li>[[gRNA Production|gRNA Production]]</li><br />
<li>[[Cocktail Protocol for Pax7|Cocktail Protocol for Pax7]]</li><br />
<br />
== Genetics ==<br />
=== Transgenic lines ===<br />
<ul><br />
<li>[[Transgenic_axolotl_lines|Axolotl (A.mexicanum)]]</li><br />
<li>[[Transgenic_frog_lines|Frog (X.laevis)]]</li><br />
</ul><br />
<br />
=== Markers ===<br />
{| class="wikitable sortable"<br />
!style="width: 150px" | Marker<br />
!style="width: 250px" | Full name<br />
!style="width: 250px" | Target<br />
!style="width: 450px" | Remarks<br />
!style="width: 200px" | Link<br />
|-<br />
|DAPI/Hoechst<br />
|4',6-diamidino-2-phenylindole<br />
|Nuclei<br />
|Incorporates into the DNA<br />
|[http://en.wikipedia.org/wiki/DAPI DAPI], [http://en.wikipedia.org/wiki/Hoechst_stain Hoechst]<br />
|-<br />
|PCNA<br />
|Proliferating-Cell-Nuclear-Antigen<br />
|Dividing cells<br />
|<br />
|[http://en.wikipedia.org/wiki/PCNA PCNA]<br />
|-<br />
|SOX2<br />
|SRY (sex determining region Y)-box 2<br />
|Preferentially neuronal lineage<br />
|Progenitor marker for both spinal cord and brain<br />
|[http://en.wikipedia.org/wiki/Sox2 SOX2]<br />
|-<br />
|PAX6<br />
|Paired box protein Pax-6<br />
|Brain/spinal cord<br />
|aka aniridia type II protein (AN2) or oculorhombin<br />
|[http://en.wikipedia.org/wiki/Pax6 PAX6]<br />
|-<br />
|PAX7<br />
|Paired box protein Pax-7<br />
|Satellite cells/spinal cord/brain<br />
|<br />
|[http://en.wikipedia.org/wiki/PAX7 PAX7]<br />
|-<br />
|MHC<br />
|Myosin heavy chain<br />
|Muscle fibers<br />
|<br />
|[http://en.wikipedia.org/wiki/Myosin_heavy_chain MHC]<br />
|-<br />
|MYF5<br />
|Myogenic factor 5<br />
|Myoblasts<br />
|Regulates muscle differentiation, labels cycling myoblasts<br />
|[http://en.wikipedia.org/wiki/Myf5 MYF5]<br />
|-<br />
|TUBB3<br />
|ßIII-tubulin<br />
|Axons<br />
|<br />
|[http://en.wikipedia.org/wiki/TUBB3 TUBB3]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Schwann cells<br />
|<br />
|-<br />
|PRRX1<br />
|Paired mesoderm homeobox protein 1<br />
|Connective tissue progenitors in Limb bud and Limb Blastema<br />
|<br />
|[http://en.wikipedia.org/wiki/PRRX1 PRRX1]<br />
|-<br />
|SMA<br />
|Smooth muscle actin<br />
|Blood vessels/endothelial cells<br />
|<br />
|<br />
|-<br />
|GFAP<br />
|Glial fibrillary acidic protein<br />
|Glia<br />
|<br />
|[http://en.wikipedia.org/wiki/Glial_fibrillary_acidic_protein GFAP]<br />
|-<br />
|NeuN<br />
|Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3<br />
|Differentiated nerve<br />
|<br />
|[http://en.wikipedia.org/wiki/NeuN NeuN]<br />
|-<br />
|LEU7/HNK1/B3GAT1/CD57<br />
|Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1<br />
|Schwann cells<br />
|<br />
|[http://en.wikipedia.org/wiki/B3GAT1 B3GAT1]<br />
|-<br />
|XBRA<br />
|Xbrachyury<br />
|Mesoderm during gastrula stage<br />
|<br />
|[http://en.wikipedia.org/wiki/Brachyury Brachyury]<br />
|-<br />
|MSX1<br />
|Muscle segment homeobox, Msh homeobox 1<br />
|Undifferentiated cells<br />
|Transcriptional repressor during embryogenesis<br />
|[http://en.wikipedia.org/wiki/MSX1 MSX1]<br />
|-<br />
|Trypan blue<br />
|<br />
|Dead tissues or cells<br />
|Vital stain<br />
|[http://en.wikipedia.org/wiki/Trypan_blue Trypan blue]<br />
|-<br />
|MEF2C<br />
|Myocyte-specific enhancer factor 2C<br />
|Differentiated muscle cells<br />
|aka MADS box transcription enhancer factor 2, polypeptide C<br />
|[http://en.wikipedia.org/wiki/Mef2c MEF2C]<br />
|-<br />
|PH3<br />
|Phospho-Histone H3<br />
|Mitotic cells<br />
|<br />
|<br />
|-<br />
|BrdU<br />
|Bromodeoxyuridine<br />
|Cells in S-phase<br />
|<br />
|[http://en.wikipedia.org/wiki/Brdu BrdU]<br />
|-<br />
|CASP3<br />
|Caspase 3<br />
|Dying cells<br />
|<br />
|[http://en.wikipedia.org/wiki/CASP3 CASP3]<br />
|-<br />
|GDF5<br />
|Growth/differentiation factor 5<br />
|Joints<br />
|Joint development marker<br />
|[http://en.wikipedia.org/wiki/GDF5 GDF5]<br />
|-<br />
|Survivin<br />
|<br />
|Cleavage furrow<br />
|aka baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5<br />
|[http://en.wikipedia.org/wiki/Survivin Survivin]<br />
|-<br />
|MAP2<br />
|Microtubule-associated protein 2<br />
|Neurons<br />
|<br />
|[http://en.wikipedia.org/wiki/MAP2 MAP2]<br />
|-<br />
|SOX9<br />
|SRY (sex determining region Y)-box 2<br />
|Cartillage progenitors<br />
|<br />
|[http://en.wikipedia.org/wiki/Sox9 SOX9]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Myelin sheath<br />
|<br />
|[http://en.wikipedia.org/wiki/Myelin_basic_protein MBP]<br />
|}<br />
<br />
== Digital data ==<br />
Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on. <br />
Select a category from the list below in order to view or download the data.<br />
<br />
=== Literature ===<br />
<ul><br />
<li>[[Literature#Theses|Theses]]</li><br />
<li>[[Literature#Publications|Publications]]</li><br />
</ul><br />
<br />
=== Databases ===<br />
<ul><br />
<li>[https://axolotl-omics.org Axolotl transcriptome] website contains the Axolotl transcriptome assemblies</li><br />
<li>[https://genome.axolotl-omics.org Axolotl genome] website contains the Axolotl genome assemblies</li><br />
<li>[http://newtomics.mpi-bn.mpg.de/ Newt transcriptome] website contains the Newt transcriptome assembly</li><br />
<li>[http://sandberg.cmb.ki.se/redspottednewt/ Red spotted newt transcriptome] website contains the red spotted newt transcriptome assembly</li><br />
<li>[https://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi Axolotl EST] database contains several ESTs and also some additional information, e.g. the position of the well with the corresponding insert in the Blastema (BL) or Neural tube (NT) library.</li><br />
<li>[http://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi?dispatch=contig_search.cgi Short insert cDNA library]</li><br />
<li>[https://axologl.axolotl-omics.org Axologle] database contains expression profiling data of Axolotl limb regeneration</li><br />
<li>[https://axologl.axolotl-omics.org/blast/blast.html Axolotl BLAST] database contains several different BLAST-able Axolotl transciptome assemblies</li><br />
</ul><br />
<br />
=== Datasets ===<br />
<ul><br />
<li>[[Datasets#Sequences|Sequences]]<br />
<ul><br />
<li>[[Datasets#Illumina|Illumina]]</li><br />
<li>[[Datasets#Sanger|Sanger]]</li><br />
<li>[[Datasets#Roche.2F454|Roche/454]]</li><br />
</ul><br />
</li><br />
<li>[[Datasets#Microarrays|Microarrays]]<br />
</ul><br />
<br />
=== Interesting links ===<br />
<ul><br />
<li>[http://www.nytimes.com/video/2013/02/27/science/100000002087758/finding-the-visible-in-the-invisible.html Visible in the invisible]: a movie introducing a new image processing ana analysis technique developed at the MIT.</li><br />
</ul></div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Axolotl&diff=859Axolotl2018-07-11T13:24:58Z<p>Sergej.nowoshilow: /* Axolotl in vitro fertilization */</p>
<hr />
<div>== Maintaining the axolotls ==<br />
== Mating ==<br />
If eggs are required for injections, it is recommended to set up mating in the afternoon (3-4 pm),<br />
#Action in the mating database must be taken first: tanaka(<i>\\storage.imp.ac.at\groups\tanaka</i>) -> Organisms -> Axolotl -> Mating History Vienna full list<br />
#Preparation of the mating tank: Choose a clean tank and check the water temperature (15-16C) at the display of the water supply.<br />
#The stock tanks with the two mating partners are put on a trolley and moved to the mating tank area.<br />
#Each animal is taken out of the tank with a net. <br />
#The transponder should be checked with the handheld scanner while the animals are in the net. The reader just responds, when it is close to the animal.<br />
#The animals are put into the mating tank. (<b>Start with the male</b>)<br />
#The handwritten ID sticker should be moved from the stock tanks to the mating tanks.<br />
#The water supply needs to be stopped by pulling up on the inlet valve until you see a yellow strip. <br />
#10-15 clean plastic leaves are put into the mating tank, swimming below the surface.<br />
#A lid is placed on top of the mating tank.<br />
#The empty stock tanks are labeled with purple tape and date.<br />
#The empty stock tanks go back to their stock position.<br />
#Poster walls are placed in front of the mating area, to protect the animals.<br />
#Axolotls don’t need to be fed during the mating.<br />
#Control after 24h, if spermatophores are visible, after 48h if eggs are visible.<br />
#Successful mating: use a bucket with tap water to put the plastic leaves in. If you want to grow the animals up, collect the eggs into a white square box and put them on the metal shelf. Not successful mating: maximal mating time should not be more than one week, otherwise animals need to be fed again.<br />
#Animals plus ID sticker are put back to the stock tanks. (Please make a check on the purple tape, if the mating was successful.)<br />
#Cleaning of mating tank and leaves: rinse the leaves carefully with tap water and put them back. Re-start circulation in the mating tank and label it with a sticker PLEASE CLEAN, then the animal caretakers take over.<br />
#Results must be reported in the database.<br />
<br />
== Feeding ==<br />
=== Artemia (Brine shrimp eggs) ===<br />
Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. <br />
<br />
Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.<br />
<br />
=== Artemia protocol (Vienna) ===<br />
# Prepare the artemia incubator funnel for hatching artemia cysts: 2 incubators per day (Saturday and Tuesday 3 incubators).<br />
# Insert heating element (adjust to 28°C).<br />
# Add 10L warm water from tap.<br />
# Wait until water is warm enough (lukewarm).<br />
# Put aerator into the funnel and start air bubbler and put lid on the top of the funnel.<br />
# Add Holtfreter´s solution: 50 ml.<br />
# Add 250g sea salt.<br />
# Wait 5 minutes so that the salt is dissolved completely.<br />
# Add 65g artemia cysts.<br />
# Hatch artemia for 48h (36-48h).<br />
# Remove lid and aerator.<br />
# Remove heating element. <br />
# There are 3 liquid phases in the funnel: 1. on top: the egg shells, 2. in the middle: nauplii (orange), 3. at the funnel outlet: unhatched cysts<br />
# Cover the upper half of the funnel with an opaque bag.<br />
# Attach a light source to the funnel outlet to attract nauplii.<br />
# Wait 5-10 minutes.<br />
# Prepare 1 x 5L-beaker, add 50ml Holtfreter´s solution and fill up with warm tap water .<br />
# Prepare 2 x empty 2L-beaker into which the nauplii have to be transferred.<br />
# Empty the funnel by opening the discharge screw on the bottom, discharge unhatched cysts (phase 3).<br />
# Collect nauplii (phase 2) in a strainer.<br />
# Discharge egg shells (phase 1).<br />
# Empty the strainer by pouring nauplii into the 2 empty beakers (equal parts).<br />
# Turn around the strainer and rinse the net with warm water from the 5L-beaker (A) so that the remaining nauplii attached to the net don´t get lost.<br />
# Fill up the 2L-beakers with warm tap water to 2L each from the 5L-beaker (A).<br />
# Wait 5-8 minutes so that the artemia phases have time to separate.<br />
# Empty the 2L-beakers into the 5L-beaker, and make sure that phases 1 (on Top) + 3 (on the bottom) are discharged (concentrate nauplii by doing so).<br />
# Add 50ml Holtfreter´s solution to both parts and fill up with warm water to 5L. <br />
# Empty the 5L-beaker with nauplii and pour nauplii into the 2 empty beakers (equal parts).<br />
# Repeat steps 24-28 as often as necessary.<br />
# Put an aerator into both beakers.<br />
<br />
Cleaning of incubators:<br />
Rinsing the funnels with cold water (do not forget the outlet connection).<br />
Clean with a soft sponge, do not scratch the plastic surface.<br />
Rinsing the funnels with hot water.<br />
<br />
=== Artemia Hatching Preparation: Protocol for 15 Ltr (Dresden). ===<br />
Switch on the bubbler. <br/ ><br />
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already. <br/ ><br />
Switch on the heating. And make sure that the light on the top is on ☺<br />
Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
=== Hatching preparation (Dresden) ===<br />
* The following protocol is considered for 15 l solution.<br />
* Switch on the bubbler.<br />
* Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.<br />
* Switch on the heating. And make sure that the light on the top is on.<br />
* Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
Keep it like this for 2 days with aeration. And >200 Lux of light.<br /><br />
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.<br /><br />
During these two days temperature can be 18°-20°C.<br /><br />
Make sure hatching container is not getting clogged during these two days.<br /><br />
<br />
== In vitro fertilization ==<br />
# Inject HCG into the dorsal muscle of the hind limb thigh of the axolotl No need to put the animals asleep, just cover the head with the wet tissue and hold them tightly while injecting.<br />
<br />
*HCG stock preparation: 10000/bottle, add 10 ml H2O to make 1000 U/ml . Freeze 1ml aliquots and keep at -20C <br />
*Inject females approx 24-26h before intended egg collection time with 350 U HCG per 100g body weight<br />
*Inject males with 200 U HCG per 100g body weight min 18-20 h before sperm collection time<br />
<br />
== Interesting observations ==<br />
Below you can find the list of observations along with the name of the person who did the observation.<br />
<br />
{| class="wikitable sortable"<br />
!style="width: 200px" | Person<br />
!style="width: 500px" class="unsortable" | Description<br />
!style="width: 50px" | Date<br />
<br />
|}</div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Axolotl&diff=857Axolotl2018-07-10T12:33:10Z<p>Sergej.nowoshilow: /* Mating */</p>
<hr />
<div>== Maintaining the axolotls ==<br />
== Mating ==<br />
If eggs are required for injections, it is recommended to set up mating in the afternoon (3-4 pm),<br />
#Action in the mating database must be taken first: tanaka(<i>\\storage.imp.ac.at\groups\tanaka</i>) -> Organisms -> Axolotl -> Mating History Vienna full list<br />
#Preparation of the mating tank: Choose a clean tank and check the water temperature (15-16C) at the display of the water supply.<br />
#The stock tanks with the two mating partners are put on a trolley and moved to the mating tank area.<br />
#Each animal is taken out of the tank with a net. <br />
#The transponder should be checked with the handheld scanner while the animals are in the net. The reader just responds, when it is close to the animal.<br />
#The animals are put into the mating tank. (<b>Start with the male</b>)<br />
#The handwritten ID sticker should be moved from the stock tanks to the mating tanks.<br />
#The water supply needs to be stopped by pulling up on the inlet valve until you see a yellow strip. <br />
#10-15 clean plastic leaves are put into the mating tank, swimming below the surface.<br />
#A lid is placed on top of the mating tank.<br />
#The empty stock tanks are labeled with purple tape and date.<br />
#The empty stock tanks go back to their stock position.<br />
#Poster walls are placed in front of the mating area, to protect the animals.<br />
#Axolotls don’t need to be fed during the mating.<br />
#Control after 24h, if spermatophores are visible, after 48h if eggs are visible.<br />
#Successful mating: use a bucket with tap water to put the plastic leaves in. If you want to grow the animals up, collect the eggs into a white square box and put them on the metal shelf. Not successful mating: maximal mating time should not be more than one week, otherwise animals need to be fed again.<br />
#Animals plus ID sticker are put back to the stock tanks. (Please make a check on the purple tape, if the mating was successful.)<br />
#Cleaning of mating tank and leaves: rinse the leaves carefully with tap water and put them back. Re-start circulation in the mating tank and label it with a sticker PLEASE CLEAN, then the animal caretakers take over.<br />
#Results must be reported in the database.<br />
<br />
== Feeding ==<br />
=== Artemia (Brine shrimp eggs) ===<br />
Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. <br />
<br />
Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.<br />
<br />
=== Artemia protocol (Vienna) ===<br />
# Prepare the artemia incubator funnel for hatching artemia cysts: 2 incubators per day (Saturday and Tuesday 3 incubators).<br />
# Insert heating element (adjust to 28°C).<br />
# Add 10L warm water from tap.<br />
# Wait until water is warm enough (lukewarm).<br />
# Put aerator into the funnel and start air bubbler and put lid on the top of the funnel.<br />
# Add Holtfreter´s solution: 50 ml.<br />
# Add 250g sea salt.<br />
# Wait 5 minutes so that the salt is dissolved completely.<br />
# Add 65g artemia cysts.<br />
# Hatch artemia for 48h (36-48h).<br />
# Remove lid and aerator.<br />
# Remove heating element. <br />
# There are 3 liquid phases in the funnel: 1. on top: the egg shells, 2. in the middle: nauplii (orange), 3. at the funnel outlet: unhatched cysts<br />
# Cover the upper half of the funnel with an opaque bag.<br />
# Attach a light source to the funnel outlet to attract nauplii.<br />
# Wait 5-10 minutes.<br />
# Prepare 1 x 5L-beaker, add 50ml Holtfreter´s solution and fill up with warm tap water .<br />
# Prepare 2 x empty 2L-beaker into which the nauplii have to be transferred.<br />
# Empty the funnel by opening the discharge screw on the bottom, discharge unhatched cysts (phase 3).<br />
# Collect nauplii (phase 2) in a strainer.<br />
# Discharge egg shells (phase 1).<br />
# Empty the strainer by pouring nauplii into the 2 empty beakers (equal parts).<br />
# Turn around the strainer and rinse the net with warm water from the 5L-beaker (A) so that the remaining nauplii attached to the net don´t get lost.<br />
# Fill up the 2L-beakers with warm tap water to 2L each from the 5L-beaker (A).<br />
# Wait 5-8 minutes so that the artemia phases have time to separate.<br />
# Empty the 2L-beakers into the 5L-beaker, and make sure that phases 1 (on Top) + 3 (on the bottom) are discharged (concentrate nauplii by doing so).<br />
# Add 50ml Holtfreter´s solution to both parts and fill up with warm water to 5L. <br />
# Empty the 5L-beaker with nauplii and pour nauplii into the 2 empty beakers (equal parts).<br />
# Repeat steps 24-28 as often as necessary.<br />
# Put an aerator into both beakers.<br />
<br />
Cleaning of incubators:<br />
Rinsing the funnels with cold water (do not forget the outlet connection).<br />
Clean with a soft sponge, do not scratch the plastic surface.<br />
Rinsing the funnels with hot water.<br />
<br />
=== Artemia Hatching Preparation: Protocol for 15 Ltr (Dresden). ===<br />
Switch on the bubbler. <br/ ><br />
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already. <br/ ><br />
Switch on the heating. And make sure that the light on the top is on ☺<br />
Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
=== Hatching preparation (Dresden) ===<br />
* The following protocol is considered for 15 l solution.<br />
* Switch on the bubbler.<br />
* Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.<br />
* Switch on the heating. And make sure that the light on the top is on.<br />
* Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
Keep it like this for 2 days with aeration. And >200 Lux of light.<br /><br />
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.<br /><br />
During these two days temperature can be 18°-20°C.<br /><br />
Make sure hatching container is not getting clogged during these two days.<br /><br />
<br />
== Interesting observations ==<br />
Below you can find the list of observations along with the name of the person who did the observation.<br />
<br />
{| class="wikitable sortable"<br />
!style="width: 200px" | Person<br />
!style="width: 500px" class="unsortable" | Description<br />
!style="width: 50px" | Date<br />
<br />
|}</div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Axolotl&diff=856Axolotl2017-08-24T12:38:43Z<p>Sergej.nowoshilow: /* Feeding */</p>
<hr />
<div>== Maintaining the axolotls ==<br />
== Mating ==<br />
If eggs are required for injections, it is recommended to set up mating in the afternoon (3-4 pm),<br />
#Action in the mating database must be taken first: tanaka(<i>\\storage.imp.ac.at\groups</i>) -> Organisms -> Axolotl -> Mating History Vienna full list<br />
#Preparation of the mating tank: Choose a clean tank and check the water temperature (15-16C) at the display of the water supply.<br />
#The stock tanks with the two mating partners are put on a trolley and moved to the mating tank area.<br />
#Each animal is taken out of the tank with a net. <br />
#The transponder should be checked with the handheld scanner while the animals are in the net. The reader just responds, when it is close to the animal.<br />
#The animals are put into the mating tank. (<b>Start with the male</b>)<br />
#The handwritten ID sticker should be moved from the stock tanks to the mating tanks.<br />
#The water supply needs to be stopped by pulling up on the inlet valve until you see a yellow strip. <br />
#10-15 clean plastic leaves are put into the mating tank, swimming below the surface.<br />
#A lid is placed on top of the mating tank.<br />
#The empty stock tanks are labeled with purple tape and date.<br />
#The empty stock tanks go back to their stock position.<br />
#Poster walls are placed in front of the mating area, to protect the animals.<br />
#Axolotls don’t need to be fed during the mating.<br />
#Control after 24h, if spermatophores are visible, after 48h if eggs are visible.<br />
#Successful mating: use a bucket with tap water to put the plastic leaves in. If you want to grow the animals up, collect the eggs into a white square box and put them on the metal shelf. Not successful mating: maximal mating time should not be more than one week, otherwise animals need to be fed again.<br />
#Animals plus ID sticker are put back to the stock tanks. (Please make a check on the purple tape, if the mating was successful.)<br />
#Cleaning of mating tank and leaves: rinse the leaves carefully with tap water and put them back. Re-start circulation in the mating tank and label it with a sticker PLEASE CLEAN, then the animal caretakers take over.<br />
#Results must be reported in the database.<br />
<br />
== Feeding ==<br />
=== Artemia (Brine shrimp eggs) ===<br />
Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. <br />
<br />
Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.<br />
<br />
=== Artemia protocol (Vienna) ===<br />
# Prepare the artemia incubator funnel for hatching artemia cysts: 2 incubators per day (Saturday and Tuesday 3 incubators).<br />
# Insert heating element (adjust to 28°C).<br />
# Add 10L warm water from tap.<br />
# Wait until water is warm enough (lukewarm).<br />
# Put aerator into the funnel and start air bubbler and put lid on the top of the funnel.<br />
# Add Holtfreter´s solution: 50 ml.<br />
# Add 250g sea salt.<br />
# Wait 5 minutes so that the salt is dissolved completely.<br />
# Add 65g artemia cysts.<br />
# Hatch artemia for 48h (36-48h).<br />
# Remove lid and aerator.<br />
# Remove heating element. <br />
# There are 3 liquid phases in the funnel: 1. on top: the egg shells, 2. in the middle: nauplii (orange), 3. at the funnel outlet: unhatched cysts<br />
# Cover the upper half of the funnel with an opaque bag.<br />
# Attach a light source to the funnel outlet to attract nauplii.<br />
# Wait 5-10 minutes.<br />
# Prepare 1 x 5L-beaker, add 50ml Holtfreter´s solution and fill up with warm tap water .<br />
# Prepare 2 x empty 2L-beaker into which the nauplii have to be transferred.<br />
# Empty the funnel by opening the discharge screw on the bottom, discharge unhatched cysts (phase 3).<br />
# Collect nauplii (phase 2) in a strainer.<br />
# Discharge egg shells (phase 1).<br />
# Empty the strainer by pouring nauplii into the 2 empty beakers (equal parts).<br />
# Turn around the strainer and rinse the net with warm water from the 5L-beaker (A) so that the remaining nauplii attached to the net don´t get lost.<br />
# Fill up the 2L-beakers with warm tap water to 2L each from the 5L-beaker (A).<br />
# Wait 5-8 minutes so that the artemia phases have time to separate.<br />
# Empty the 2L-beakers into the 5L-beaker, and make sure that phases 1 (on Top) + 3 (on the bottom) are discharged (concentrate nauplii by doing so).<br />
# Add 50ml Holtfreter´s solution to both parts and fill up with warm water to 5L. <br />
# Empty the 5L-beaker with nauplii and pour nauplii into the 2 empty beakers (equal parts).<br />
# Repeat steps 24-28 as often as necessary.<br />
# Put an aerator into both beakers.<br />
<br />
Cleaning of incubators:<br />
Rinsing the funnels with cold water (do not forget the outlet connection).<br />
Clean with a soft sponge, do not scratch the plastic surface.<br />
Rinsing the funnels with hot water.<br />
<br />
=== Artemia Hatching Preparation: Protocol for 15 Ltr (Dresden). ===<br />
Switch on the bubbler. <br/ ><br />
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already. <br/ ><br />
Switch on the heating. And make sure that the light on the top is on ☺<br />
Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
=== Hatching preparation (Dresden) ===<br />
* The following protocol is considered for 15 l solution.<br />
* Switch on the bubbler.<br />
* Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.<br />
* Switch on the heating. And make sure that the light on the top is on.<br />
* Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
Keep it like this for 2 days with aeration. And >200 Lux of light.<br /><br />
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.<br /><br />
During these two days temperature can be 18°-20°C.<br /><br />
Make sure hatching container is not getting clogged during these two days.<br /><br />
<br />
== Interesting observations ==<br />
Below you can find the list of observations along with the name of the person who did the observation.<br />
<br />
{| class="wikitable sortable"<br />
!style="width: 200px" | Person<br />
!style="width: 500px" class="unsortable" | Description<br />
!style="width: 50px" | Date<br />
<br />
|}</div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Axolotl&diff=855Axolotl2017-08-23T14:08:47Z<p>Sergej.nowoshilow: </p>
<hr />
<div>== Maintaining the axolotls ==<br />
== Mating ==<br />
If eggs are required for injections, it is recommended to set up mating in the afternoon (3-4 pm),<br />
#Action in the mating database must be taken first: tanaka(<i>\\storage.imp.ac.at\groups</i>) -> Organisms -> Axolotl -> Mating History Vienna full list<br />
#Preparation of the mating tank: Choose a clean tank and check the water temperature (15-16C) at the display of the water supply.<br />
#The stock tanks with the two mating partners are put on a trolley and moved to the mating tank area.<br />
#Each animal is taken out of the tank with a net. <br />
#The transponder should be checked with the handheld scanner while the animals are in the net. The reader just responds, when it is close to the animal.<br />
#The animals are put into the mating tank. (<b>Start with the male</b>)<br />
#The handwritten ID sticker should be moved from the stock tanks to the mating tanks.<br />
#The water supply needs to be stopped by pulling up on the inlet valve until you see a yellow strip. <br />
#10-15 clean plastic leaves are put into the mating tank, swimming below the surface.<br />
#A lid is placed on top of the mating tank.<br />
#The empty stock tanks are labeled with purple tape and date.<br />
#The empty stock tanks go back to their stock position.<br />
#Poster walls are placed in front of the mating area, to protect the animals.<br />
#Axolotls don’t need to be fed during the mating.<br />
#Control after 24h, if spermatophores are visible, after 48h if eggs are visible.<br />
#Successful mating: use a bucket with tap water to put the plastic leaves in. If you want to grow the animals up, collect the eggs into a white square box and put them on the metal shelf. Not successful mating: maximal mating time should not be more than one week, otherwise animals need to be fed again.<br />
#Animals plus ID sticker are put back to the stock tanks. (Please make a check on the purple tape, if the mating was successful.)<br />
#Cleaning of mating tank and leaves: rinse the leaves carefully with tap water and put them back. Re-start circulation in the mating tank and label it with a sticker PLEASE CLEAN, then the animal caretakers take over.<br />
#Results must be reported in the database.<br />
<br />
== Feeding ==<br />
=== Artemia (Brine shrimp eggs) ===<br />
Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. <br />
<br />
Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.<br />
<br />
=== Artemia Hatching Preparation: Protocol for 15 Ltr. ===<br />
Switch on the bubbler. <br/ ><br />
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already. <br/ ><br />
Switch on the heating. And make sure that the light on the top is on ☺<br />
Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
=== Hatching preparation ===<br />
* The following protocol is considered for 15 l solution.<br />
* Switch on the bubbler.<br />
* Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.<br />
* Switch on the heating. And make sure that the light on the top is on.<br />
* Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
Keep it like this for 2 days with aeration. And >200 Lux of light.<br /><br />
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.<br /><br />
During these two days temperature can be 18°-20°C.<br /><br />
Make sure hatching container is not getting clogged during these two days.<br /><br />
<br />
== Interesting observations ==<br />
Below you can find the list of observations along with the name of the person who did the observation.<br />
<br />
{| class="wikitable sortable"<br />
!style="width: 200px" | Person<br />
!style="width: 500px" class="unsortable" | Description<br />
!style="width: 50px" | Date<br />
<br />
|}</div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Axolotl&diff=854Axolotl2017-08-23T14:03:54Z<p>Sergej.nowoshilow: </p>
<hr />
<div>== Maintaining the axolotls ==<br />
== Mating ==<br />
bla<br />
== Feeding ==<br />
=== Artemia (Brine shrimp eggs) ===<br />
Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. <br />
<br />
Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.<br />
<br />
=== Artemia Hatching Preparation: Protocol for 15 Ltr. ===<br />
Switch on the bubbler. <br/ ><br />
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already. <br/ ><br />
Switch on the heating. And make sure that the light on the top is on ☺<br />
Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
=== Hatching preparation ===<br />
* The following protocol is considered for 15 l solution.<br />
* Switch on the bubbler.<br />
* Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.<br />
* Switch on the heating. And make sure that the light on the top is on.<br />
* Add 50-60 ml full of dried artemia (Stored at 40C) to this.<br />
<br />
Keep it like this for 2 days with aeration. And >200 Lux of light.<br /><br />
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.<br /><br />
During these two days temperature can be 18°-20°C.<br /><br />
Make sure hatching container is not getting clogged during these two days.<br /><br />
<br />
== Interesting observations ==<br />
Below you can find the list of observations along with the name of the person who did the observation.<br />
<br />
{| class="wikitable sortable"<br />
!style="width: 200px" | Person<br />
!style="width: 500px" class="unsortable" | Description<br />
!style="width: 50px" | Date<br />
<br />
|}</div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Overview&diff=853Overview2017-08-23T14:03:27Z<p>Sergej.nowoshilow: /* Mating */</p>
<hr />
<div>== Introduction ==<br />
On the pages listed below you can find most common protocols used in the lab as well as a list of lab duties along with the names of the responsible persons. Please, contact the responsible persons if you have any question, suggestions or troubles.<br />
<br />
== Axolotl ==<br />
<br />
=== Maintainance ===<br />
In this section you will find some general information on how to [[Axolotl#Maintaining_the_axolotls|maintain]] the animals.<br />
<br />
=== Mating ===<br />
In this section you will find the information on how to [[Axolotl#Mating_the_axolotls|mate]] axolotls.<br />
<br />
=== Feeding ===<br />
This section will give you an overview of how to [[Axolotl#Feeding|feed]] the axolotls.<br />
<br />
=== Interesting observations ===<br />
In the following section you can find some [[Axolotl#Interesting_observations|interesting observations]] concerning the axolotl, its behavior, caveats of the transgenics and many more.<br />
<br />
== Lab duties ==<br />
<ul><br />
=== Chemicals ===<br />
<li>[[Antibiotics|Antibiotics]]</li><br />
<li>[[Benzocaine|Benzocaine]]</li><br />
<li>[[Fibronectin|Fibronectin]]</li><br />
<li>[[Gelatine|Gelatine]]</li><br />
<li>[[Liquid_nitrogen|Liquid nitrogen]]</li><br />
<li>[[Primary_antibodies|Primary antibodies]]</li><br />
<li>[[Secondary_antibodies|Secondary antibodies]]</li><br />
<li>[[Serum_heat_inactivation|Serum heat inactivation]]</li><br />
<br />
=== Hardware ===<br />
<li>[[Autoclave|Autoclave]]</li><br />
<li>[[Cryostat|Cryostat]]</li><br />
<li>[[Dissecting_microscopes|Dissecting microscopes]]</li><br />
<li>[[Leica_confocal_microscope|Leica confocal microscope]]</li><br />
<li>[[Vacuum_pumps|Vacuum pumps]]</li><br />
<li>[[Waterbath|Waterbath]]</li><br />
<br />
=== General ===<br />
<li>[[Ordering_common_stuff|Ordering common stuff]]</li><br />
<li>[[Heat_shock_competent_cells|Heat-shock competent cells]]</li><br />
</ul><br />
<br />
<br />
== Protocols ==<br />
<ul><br />
=== Cell culture ===<br />
<li>[[Adhesive_cell_culture|Adhesive cell culture]]</li><br />
<li>[[Baculovirus_titration|Baculovirus titration]]</li><br />
<li>[[Cardiomyocyte_Preparation|Cardiomyocyte preparation]]</li><br />
<li>[[Electroporation_of_nec|Electroporation of neural epithelia cells (axolotl spinal cord)]]</li><br />
<li>[[Myoblasts_electroporation|Myoblasts electroporation]]</li><br />
<li>[[Neurosphere_culture|Neurosphere culture]]</li><br />
<li>[[Thawing_293FT_cells|Thawing 293FT cells]]</li><br />
<li>[[G418_medium_preparation|G418 medium preparation]]</li><br />
<br />
===== A1 cells =====<br />
<br />
<li>[[Thawing_frozen_A1_cells|Thawing frozen A1 cells]]</li><br />
<li>[[Freezing_of_A1_cells|Freezing of A1 cells]]</li><br />
<li>[[Passaging_A1_cell|A1 cell passaging]]</li><br />
<li>[[A1_cell_passaging_myotube|A1 cell passaging for myotube preparation]]</li><br />
<li>[[Modified_myotube_prep|Modified myotube prep]]</li><br />
<li>[[A1_cell_transfection_with_FuGene|A1 cell transfection with FuGene]]</li><br />
<li>[[Electroporation_of_A1_cells|Electroporation of A1 cells]]</li><br />
<li>[[BrdU_labelling_of_A1_cells_in_96-well_plate|BrdU labelling of A1 cells in 96-well plate]]</li><br />
<li>[[BrdU_and_myosin_staining|BrdU and myosin staining]]</li><br />
<li>[[Preparation_of_Low_Serum_for_A1_cells|Preparation of Low Serum for A1 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_A1_cells|Preparation of High Serum for A1 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_A1_cells|Preparation of Freezing media for A1 cells]]</li><br />
<br />
===== Blastema cells =====<br />
<li>[[Blastema_cells|Blastema cells]]</li><br />
<li>[[Electroporation_of_blastema_cells|Electroporation of blastema cells]]</li><br />
<br />
===== C2C12 cells =====<br />
<br />
<li>[[Thawing_frozen_C2C12_cells|Thawing frozen C2C12 cells]]</li><br />
<li>[[Freezing_of_C2C12_cells|Freezing of C2C12 cells]]</li><br />
<li>[[Passaging_C2C12_cells|C2C12 cell passaging]]</li><br />
<li>[[Myotube_preparation_of_C2C12_cells|Myotube preparation of C2C12 cells]]</li><br />
<li>[[C2C12_cell_transfection_with_FuGene|C2C12 cell transfection with FuGene]]</li><br />
<li>[[Cloning_of_C2C12_cells|Cloning of C2C12 cells]]</li><br />
<li>[[Electroporation_of_C2C12_cells|Electroporation of C2C12 cells]]</li><br />
<li>[[Preparation_of_Low_Serum_for_C2C12_cells|Preparation of Low Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_C2C12_cells|Preparation of High Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_C2C12_cells|Preparation of Freezing media for C2C12 cells]]</li><br />
<br />
===== Embryonic stem cells =====<br />
<li>[[mESC_culture|mESC culture/Cyst formation]]</li><br />
<br />
===== Hybridoma cells =====<br />
<li>[[Thawing_(XB10)_hybridoma_cells|Thawing (XB10) hybridoma cells]]</li><br />
<li>[[Hybridoma_cells_in_SFX_media|Hybridoma cells in SFX media]]</li><br />
<li>[[Hybridoma_cells_clones|Hybridoma cell clones]]</li><br />
<br />
=== Histology ===<br />
<li>[[Alcian_Blue_Staining|Alcian Blue Staining]]</li><br />
<li>[[Double_Immunostaining_via_antigen_retrieval|Double Immunostaining via antigen retrieval (Sherry)]]</li><br />
<li>[[TSA_Staining_on_axolotl-tissue|Tyramide signal amplification (TSA) staining on axolotl tissue]]</li><br />
<br />
===== In situ Hybridization =====<br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Anja)]]</li><br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Akira)]]</li><br />
<li>[[Whole_mount_ISH_on_axolotl_tissue|Whole mount ISH on axolotl tissue]]</li><br />
<br />
=== Antibody ===<br />
===== Monoclonal Antibodies: =====<br />
<li>[[Purifying IgG1 from Hybridoma Supernatant]]</li><br />
<li>[[Labelling myosin antibody]]</li><br />
<li>[[Labelling Pax6 with Dig-NHS]]</li><br />
<li>[[Anti-BrDU Antibody labeling]]</li><br />
<br />
===== Polyclonal Antibodies: =====<br />
<li>[[Address for making polyclonal antibodies]]</li><br />
<li>[[Letter to Froppier (French)]]</li><br />
<li>[[labeled AB staining]]</li><br />
<li>[[Antibody staining]]</li><br />
<br />
=== Molecular biology ===<br />
<li>[[Metamorphosis_protocol|Metamorphosis protocol]]</li><br />
<li>[[Digestion_DNA|Plasmid digestion]]</li><br />
<li>[[RNA_Extraction_from_axolotl_tissue|RNA Extraction (axolotl tissue)]]</li><br />
<li>[[RNA_formaldehyde_gels|RNA formaldehyde gels]]</li><br />
<br />
== Lab business ==<br />
=== Ordering database ===<br />
Use the following [http://ordering-db.biotec.tu-dresden.de/ordering/login.gsp ordering database] to place your orders.<br />
<br />
=== Booking calendars ===<br />
Since some pieces of hardware are used intensively it is necessary to book them in advance. The links to the corresponding booking calendars are listed below.<br />
<ul><br />
<li><br />
Fluorescence microscopes<br />
<ul><br />
<li>[http://www.my.calendars.net/olympusszx16 Olympus SZX 16]</li><br />
<li>[http://www.my.calendars.net/new_olympus/ New Olympus SZX 16]</li><br />
<li>[http://www.my.calendars.net/Olympus_OVK Olympus OVK]</li><br />
<li>[http://www.my.calendars.net/axio_observer/ Axio Observer]</li><br />
</ul><br />
</li><br />
<li><br />
Microinjectors and electroporators and dissecting microscope<br />
<ul><br />
<li>Room 0.232<br />
<ul> <br />
<li>[http://www.my.calendars.net/electroporator Nepa Gene]</li><br />
<li>[http://www.my.calendars.net/electroporator2 ECM 830]</li><br />
<li>[http://www.my.calendars.net/stereoscope2/d01/11/2012 Olympus dissecting microscope]</li><br />
</ul><br />
</li><br />
<li>Lab<br />
<ul><br />
<li>[http://www.my.calendars.net/OlympusLab Olympus (next to the axolotls)]</li><br />
<li>[http://www.my.calendars.net/OlympusLab2 Olympus (next to the Microtome)]</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
<li><br />
Confocal microscopes<br />
<ul><br />
<li>[http://www.my.calendars.net/leica_lsm Leica LSM]</li><br />
</ul><br />
</li><br />
<li><br />
Cryostats<br />
<ul><br />
<li>[http://www.my.calendars.net/cryostat1 Cryostat 1]</li><br />
<li>[http://www.my.calendars.net/cryostat2 Cryostat 2]</li><br />
</ul><br />
</li><br />
<li><br />
Microtome<br />
<ul><br />
<li>[http://www.my.calendars.net/microtome Microtome]</li><br />
</ul><br />
</li><br />
<li><br />
Paraffin embedding station<br />
<ul><br />
<li>[http://www.my.calendars.net/wax_station Paraffin embedding station]</li><br />
</ul><br />
</li><br />
</ul><br />
<br />
== Genetics ==<br />
=== Transgenic lines ===<br />
<ul><br />
<li>[[Transgenic_axolotl_lines|Axolotl (A.mexicanum)]]</li><br />
<li>[[Transgenic_frog_lines|Frog (X.laevis)]]</li><br />
</ul><br />
<br />
=== Markers ===<br />
{| class="wikitable sortable"<br />
!style="width: 150px" | Marker<br />
!style="width: 250px" | Full name<br />
!style="width: 250px" | Target<br />
!style="width: 450px" | Remarks<br />
!style="width: 200px" | Link<br />
|-<br />
|DAPI/Hoechst<br />
|4',6-diamidino-2-phenylindole<br />
|Nuclei<br />
|Incorporates into the DNA<br />
|[http://en.wikipedia.org/wiki/DAPI DAPI], [http://en.wikipedia.org/wiki/Hoechst_stain Hoechst]<br />
|-<br />
|PCNA<br />
|Proliferating-Cell-Nuclear-Antigen<br />
|Dividing cells<br />
|<br />
|[http://en.wikipedia.org/wiki/PCNA PCNA]<br />
|-<br />
|SOX2<br />
|SRY (sex determining region Y)-box 2<br />
|Preferentially neuronal lineage<br />
|Progenitor marker for both spinal cord and brain<br />
|[http://en.wikipedia.org/wiki/Sox2 SOX2]<br />
|-<br />
|PAX6<br />
|Paired box protein Pax-6<br />
|Brain/spinal cord<br />
|aka aniridia type II protein (AN2) or oculorhombin<br />
|[http://en.wikipedia.org/wiki/Pax6 PAX6]<br />
|-<br />
|PAX7<br />
|Paired box protein Pax-7<br />
|Satellite cells/spinal cord/brain<br />
|<br />
|[http://en.wikipedia.org/wiki/PAX7 PAX7]<br />
|-<br />
|MHC<br />
|Myosin heavy chain<br />
|Muscle fibers<br />
|<br />
|[http://en.wikipedia.org/wiki/Myosin_heavy_chain MHC]<br />
|-<br />
|MYF5<br />
|Myogenic factor 5<br />
|Myoblasts<br />
|Regulates muscle differentiation, labels cycling myoblasts<br />
|[http://en.wikipedia.org/wiki/Myf5 MYF5]<br />
|-<br />
|TUBB3<br />
|ßIII-tubulin<br />
|Axons<br />
|<br />
|[http://en.wikipedia.org/wiki/TUBB3 TUBB3]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Schwann cells<br />
|<br />
|-<br />
|PRRX1<br />
|Paired mesoderm homeobox protein 1<br />
|Connective tissue progenitors in Limb bud and Limb Blastema<br />
|<br />
|[http://en.wikipedia.org/wiki/PRRX1 PRRX1]<br />
|-<br />
|SMA<br />
|Smooth muscle actin<br />
|Blood vessels/endothelial cells<br />
|<br />
|<br />
|-<br />
|GFAP<br />
|Glial fibrillary acidic protein<br />
|Glia<br />
|<br />
|[http://en.wikipedia.org/wiki/Glial_fibrillary_acidic_protein GFAP]<br />
|-<br />
|NeuN<br />
|Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3<br />
|Differentiated nerve<br />
|<br />
|[http://en.wikipedia.org/wiki/NeuN NeuN]<br />
|-<br />
|LEU7/HNK1/B3GAT1/CD57<br />
|Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1<br />
|Schwann cells<br />
|<br />
|[http://en.wikipedia.org/wiki/B3GAT1 B3GAT1]<br />
|-<br />
|XBRA<br />
|Xbrachyury<br />
|Mesoderm during gastrula stage<br />
|<br />
|[http://en.wikipedia.org/wiki/Brachyury Brachyury]<br />
|-<br />
|MSX1<br />
|Muscle segment homeobox, Msh homeobox 1<br />
|Undifferentiated cells<br />
|Transcriptional repressor during embryogenesis<br />
|[http://en.wikipedia.org/wiki/MSX1 MSX1]<br />
|-<br />
|Trypan blue<br />
|<br />
|Dead tissues or cells<br />
|Vital stain<br />
|[http://en.wikipedia.org/wiki/Trypan_blue Trypan blue]<br />
|-<br />
|MEF2C<br />
|Myocyte-specific enhancer factor 2C<br />
|Differentiated muscle cells<br />
|aka MADS box transcription enhancer factor 2, polypeptide C<br />
|[http://en.wikipedia.org/wiki/Mef2c MEF2C]<br />
|-<br />
|PH3<br />
|Phospho-Histone H3<br />
|Mitotic cells<br />
|<br />
|<br />
|-<br />
|BrdU<br />
|Bromodeoxyuridine<br />
|Cells in S-phase<br />
|<br />
|[http://en.wikipedia.org/wiki/Brdu BrdU]<br />
|-<br />
|CASP3<br />
|Caspase 3<br />
|Dying cells<br />
|<br />
|[http://en.wikipedia.org/wiki/CASP3 CASP3]<br />
|-<br />
|GDF5<br />
|Growth/differentiation factor 5<br />
|Joints<br />
|Joint development marker<br />
|[http://en.wikipedia.org/wiki/GDF5 GDF5]<br />
|-<br />
|Survivin<br />
|<br />
|Cleavage furrow<br />
|aka baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5<br />
|[http://en.wikipedia.org/wiki/Survivin Survivin]<br />
|-<br />
|MAP2<br />
|Microtubule-associated protein 2<br />
|Neurons<br />
|<br />
|[http://en.wikipedia.org/wiki/MAP2 MAP2]<br />
|-<br />
|SOX9<br />
|SRY (sex determining region Y)-box 2<br />
|Cartillage progenitors<br />
|<br />
|[http://en.wikipedia.org/wiki/Sox9 SOX9]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Myelin sheath<br />
|<br />
|[http://en.wikipedia.org/wiki/Myelin_basic_protein MBP]<br />
|}<br />
<br />
== Digital data ==<br />
Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on. <br />
Select a category from the list below in order to view or download the data.<br />
<br />
=== Fileservers ===<br />
{| class="wikitable"<br />
!style="width: 350px" | Location<br />
!style="width: 250px" | Volume size<br />
!style="width: 350px" | Description<br />
!style="width: 350px" | Access<br />
!style="width: 350px" | Location (console only)<br />
|-<br />
|//biodata/groups/<b>crtd_tanaka</b><br />
|16 TB<br />
|General purpose storage space<br />
|Entire Tanaka lab<br />
|<i>biocluster:</i> <b>/group/crtd_tanaka</b><br />
|-<br />
|//biodata/groups/<b>tanaka-ests</b><br />
|247 GB<br />
|EST sequences<br />
|Entire Tanaka lab<br />
|<i>biocluster:</i> <b>/group/tanaka-ests</b><br />
|-<br />
|//biodata/groups/<b>axolotl-transcriptome</b><br />
|500 GB<br />
|Axolotl transcriptome data<br />
|Elly, Sergej<br />
|<i>biocluster:</i> <b>/projects/axolotl-transcriptome</b><br />
|-<br />
|//biodata/groups/<b>tanaka-saori</b><br />
|878 GB<br />
|Special directory for Saori's data<br />
|Elly, Saori, Sergej<br />
|<i>biocluster:</i> <b>/group/tanaka-saori</b><br />
|}<br />
<br />
{{Tip|The path is different, when accessing the file server from the console.}}<br />
<br />
=== Literature ===<br />
<ul><br />
<li>[[Literature#Theses|Theses]]</li><br />
<li>[[Literature#Publications|Publications]]</li><br />
</ul><br />
<br />
=== Databases ===<br />
<ul><br />
<li>[http://elbrus.biochem.mpg.de/ Axolotl transcriptome] website contains the Axolotl transcriptome assemblies</li><br />
<li>[http://newtomics.mpi-bn.mpg.de/ Newt transcriptome] website contains the Newt transcriptome assembly</li><br />
<li>[http://sandberg.cmb.ki.se/redspottednewt/ Red spotted newt transcriptome] website contains the red spotted newt transcriptome assembly</li><br />
<li>[https://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi Axolotl EST] database contains several ESTs and also some additional information, e.g. the position of the well with the corresponding insert in the Blastema (BL) or Neural tube (NT) library.</li><br />
<li>[http://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi?dispatch=contig_search.cgi Short insert cDNA library]</li><br />
<li>CURRENTLY UNAVAILABLE! [http://est.age.mpg.de/ Axologle] database contains expression profiling data of Axolotl limb regeneration</li><br />
<li>CURRENTLY UNAVAILABLE! [http://est.age.mpg.de/blast/blast.html Axolotl BLAST] database contains several different BLAST-able Axolotl transciptome assemblies</li><br />
</ul><br />
<br />
=== Datasets ===<br />
<ul><br />
<li>[[Datasets#Sequences|Sequences]]<br />
<ul><br />
<li>[[Datasets#Illumina|Illumina]]</li><br />
<li>[[Datasets#Sanger|Sanger]]</li><br />
<li>[[Datasets#Roche.2F454|Roche/454]]</li><br />
</ul><br />
</li><br />
<li>[[Datasets#Microarrays|Microarrays]]<br />
</ul><br />
<br />
=== Interesting links ===<br />
<ul><br />
<li>[http://www.nytimes.com/video/2013/02/27/science/100000002087758/finding-the-visible-in-the-invisible.html Visible in the invisible]: a movie introducing a new image processing ana analysis technique developed at the MIT.</li><br />
</ul><br />
<br />
== Lab life ==<br />
<ul><br />
<li>[[Lab_photos|Lab photos]]</li><br />
<li>Further pictures can be found in <b>/biodata/groups/crtd_tanaka/LabFun/</b></li><br />
<li>The recepies for Heino's farewell present can be found under <b>/biodata/groups/crtd_tanaka/LabFun/Heino's farewell present/</b></li><br />
<li>The video of the visit of the EU commissioner can be found under <b>/biodata/groups/crtd_tanaka/LabFun/</b></li><br />
</ul></div>Sergej.nowoshilowhttps://wiki.tanakalab.org/index.php?title=Overview&diff=852Overview2017-08-23T14:02:34Z<p>Sergej.nowoshilow: /* Axolotl */</p>
<hr />
<div>== Introduction ==<br />
On the pages listed below you can find most common protocols used in the lab as well as a list of lab duties along with the names of the responsible persons. Please, contact the responsible persons if you have any question, suggestions or troubles.<br />
<br />
== Axolotl ==<br />
<br />
=== Maintainance ===<br />
In this section you will find some general information on how to [[Axolotl#Maintaining_the_axolotls|maintain]] the animals.<br />
<br />
=== Mating ===<br />
In this section you will find the information on how to [[Axolotl#Maintaining_the_axolotls|mate]] axolotls.<br />
<br />
=== Feeding ===<br />
This section will give you an overview of how to [[Axolotl#Feeding|feed]] the axolotls.<br />
<br />
=== Interesting observations ===<br />
In the following section you can find some [[Axolotl#Interesting_observations|interesting observations]] concerning the axolotl, its behavior, caveats of the transgenics and many more.<br />
<br />
== Lab duties ==<br />
<ul><br />
=== Chemicals ===<br />
<li>[[Antibiotics|Antibiotics]]</li><br />
<li>[[Benzocaine|Benzocaine]]</li><br />
<li>[[Fibronectin|Fibronectin]]</li><br />
<li>[[Gelatine|Gelatine]]</li><br />
<li>[[Liquid_nitrogen|Liquid nitrogen]]</li><br />
<li>[[Primary_antibodies|Primary antibodies]]</li><br />
<li>[[Secondary_antibodies|Secondary antibodies]]</li><br />
<li>[[Serum_heat_inactivation|Serum heat inactivation]]</li><br />
<br />
=== Hardware ===<br />
<li>[[Autoclave|Autoclave]]</li><br />
<li>[[Cryostat|Cryostat]]</li><br />
<li>[[Dissecting_microscopes|Dissecting microscopes]]</li><br />
<li>[[Leica_confocal_microscope|Leica confocal microscope]]</li><br />
<li>[[Vacuum_pumps|Vacuum pumps]]</li><br />
<li>[[Waterbath|Waterbath]]</li><br />
<br />
=== General ===<br />
<li>[[Ordering_common_stuff|Ordering common stuff]]</li><br />
<li>[[Heat_shock_competent_cells|Heat-shock competent cells]]</li><br />
</ul><br />
<br />
<br />
== Protocols ==<br />
<ul><br />
=== Cell culture ===<br />
<li>[[Adhesive_cell_culture|Adhesive cell culture]]</li><br />
<li>[[Baculovirus_titration|Baculovirus titration]]</li><br />
<li>[[Cardiomyocyte_Preparation|Cardiomyocyte preparation]]</li><br />
<li>[[Electroporation_of_nec|Electroporation of neural epithelia cells (axolotl spinal cord)]]</li><br />
<li>[[Myoblasts_electroporation|Myoblasts electroporation]]</li><br />
<li>[[Neurosphere_culture|Neurosphere culture]]</li><br />
<li>[[Thawing_293FT_cells|Thawing 293FT cells]]</li><br />
<li>[[G418_medium_preparation|G418 medium preparation]]</li><br />
<br />
===== A1 cells =====<br />
<br />
<li>[[Thawing_frozen_A1_cells|Thawing frozen A1 cells]]</li><br />
<li>[[Freezing_of_A1_cells|Freezing of A1 cells]]</li><br />
<li>[[Passaging_A1_cell|A1 cell passaging]]</li><br />
<li>[[A1_cell_passaging_myotube|A1 cell passaging for myotube preparation]]</li><br />
<li>[[Modified_myotube_prep|Modified myotube prep]]</li><br />
<li>[[A1_cell_transfection_with_FuGene|A1 cell transfection with FuGene]]</li><br />
<li>[[Electroporation_of_A1_cells|Electroporation of A1 cells]]</li><br />
<li>[[BrdU_labelling_of_A1_cells_in_96-well_plate|BrdU labelling of A1 cells in 96-well plate]]</li><br />
<li>[[BrdU_and_myosin_staining|BrdU and myosin staining]]</li><br />
<li>[[Preparation_of_Low_Serum_for_A1_cells|Preparation of Low Serum for A1 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_A1_cells|Preparation of High Serum for A1 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_A1_cells|Preparation of Freezing media for A1 cells]]</li><br />
<br />
===== Blastema cells =====<br />
<li>[[Blastema_cells|Blastema cells]]</li><br />
<li>[[Electroporation_of_blastema_cells|Electroporation of blastema cells]]</li><br />
<br />
===== C2C12 cells =====<br />
<br />
<li>[[Thawing_frozen_C2C12_cells|Thawing frozen C2C12 cells]]</li><br />
<li>[[Freezing_of_C2C12_cells|Freezing of C2C12 cells]]</li><br />
<li>[[Passaging_C2C12_cells|C2C12 cell passaging]]</li><br />
<li>[[Myotube_preparation_of_C2C12_cells|Myotube preparation of C2C12 cells]]</li><br />
<li>[[C2C12_cell_transfection_with_FuGene|C2C12 cell transfection with FuGene]]</li><br />
<li>[[Cloning_of_C2C12_cells|Cloning of C2C12 cells]]</li><br />
<li>[[Electroporation_of_C2C12_cells|Electroporation of C2C12 cells]]</li><br />
<li>[[Preparation_of_Low_Serum_for_C2C12_cells|Preparation of Low Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_High_Serum_for_C2C12_cells|Preparation of High Serum for C2C12 cells]]</li><br />
<li>[[Preparation_of_Freezing_media_for_C2C12_cells|Preparation of Freezing media for C2C12 cells]]</li><br />
<br />
===== Embryonic stem cells =====<br />
<li>[[mESC_culture|mESC culture/Cyst formation]]</li><br />
<br />
===== Hybridoma cells =====<br />
<li>[[Thawing_(XB10)_hybridoma_cells|Thawing (XB10) hybridoma cells]]</li><br />
<li>[[Hybridoma_cells_in_SFX_media|Hybridoma cells in SFX media]]</li><br />
<li>[[Hybridoma_cells_clones|Hybridoma cell clones]]</li><br />
<br />
=== Histology ===<br />
<li>[[Alcian_Blue_Staining|Alcian Blue Staining]]</li><br />
<li>[[Double_Immunostaining_via_antigen_retrieval|Double Immunostaining via antigen retrieval (Sherry)]]</li><br />
<li>[[TSA_Staining_on_axolotl-tissue|Tyramide signal amplification (TSA) staining on axolotl tissue]]</li><br />
<br />
===== In situ Hybridization =====<br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Anja)]]</li><br />
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Akira)]]</li><br />
<li>[[Whole_mount_ISH_on_axolotl_tissue|Whole mount ISH on axolotl tissue]]</li><br />
<br />
=== Antibody ===<br />
===== Monoclonal Antibodies: =====<br />
<li>[[Purifying IgG1 from Hybridoma Supernatant]]</li><br />
<li>[[Labelling myosin antibody]]</li><br />
<li>[[Labelling Pax6 with Dig-NHS]]</li><br />
<li>[[Anti-BrDU Antibody labeling]]</li><br />
<br />
===== Polyclonal Antibodies: =====<br />
<li>[[Address for making polyclonal antibodies]]</li><br />
<li>[[Letter to Froppier (French)]]</li><br />
<li>[[labeled AB staining]]</li><br />
<li>[[Antibody staining]]</li><br />
<br />
=== Molecular biology ===<br />
<li>[[Metamorphosis_protocol|Metamorphosis protocol]]</li><br />
<li>[[Digestion_DNA|Plasmid digestion]]</li><br />
<li>[[RNA_Extraction_from_axolotl_tissue|RNA Extraction (axolotl tissue)]]</li><br />
<li>[[RNA_formaldehyde_gels|RNA formaldehyde gels]]</li><br />
<br />
== Lab business ==<br />
=== Ordering database ===<br />
Use the following [http://ordering-db.biotec.tu-dresden.de/ordering/login.gsp ordering database] to place your orders.<br />
<br />
=== Booking calendars ===<br />
Since some pieces of hardware are used intensively it is necessary to book them in advance. The links to the corresponding booking calendars are listed below.<br />
<ul><br />
<li><br />
Fluorescence microscopes<br />
<ul><br />
<li>[http://www.my.calendars.net/olympusszx16 Olympus SZX 16]</li><br />
<li>[http://www.my.calendars.net/new_olympus/ New Olympus SZX 16]</li><br />
<li>[http://www.my.calendars.net/Olympus_OVK Olympus OVK]</li><br />
<li>[http://www.my.calendars.net/axio_observer/ Axio Observer]</li><br />
</ul><br />
</li><br />
<li><br />
Microinjectors and electroporators and dissecting microscope<br />
<ul><br />
<li>Room 0.232<br />
<ul> <br />
<li>[http://www.my.calendars.net/electroporator Nepa Gene]</li><br />
<li>[http://www.my.calendars.net/electroporator2 ECM 830]</li><br />
<li>[http://www.my.calendars.net/stereoscope2/d01/11/2012 Olympus dissecting microscope]</li><br />
</ul><br />
</li><br />
<li>Lab<br />
<ul><br />
<li>[http://www.my.calendars.net/OlympusLab Olympus (next to the axolotls)]</li><br />
<li>[http://www.my.calendars.net/OlympusLab2 Olympus (next to the Microtome)]</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
<li><br />
Confocal microscopes<br />
<ul><br />
<li>[http://www.my.calendars.net/leica_lsm Leica LSM]</li><br />
</ul><br />
</li><br />
<li><br />
Cryostats<br />
<ul><br />
<li>[http://www.my.calendars.net/cryostat1 Cryostat 1]</li><br />
<li>[http://www.my.calendars.net/cryostat2 Cryostat 2]</li><br />
</ul><br />
</li><br />
<li><br />
Microtome<br />
<ul><br />
<li>[http://www.my.calendars.net/microtome Microtome]</li><br />
</ul><br />
</li><br />
<li><br />
Paraffin embedding station<br />
<ul><br />
<li>[http://www.my.calendars.net/wax_station Paraffin embedding station]</li><br />
</ul><br />
</li><br />
</ul><br />
<br />
== Genetics ==<br />
=== Transgenic lines ===<br />
<ul><br />
<li>[[Transgenic_axolotl_lines|Axolotl (A.mexicanum)]]</li><br />
<li>[[Transgenic_frog_lines|Frog (X.laevis)]]</li><br />
</ul><br />
<br />
=== Markers ===<br />
{| class="wikitable sortable"<br />
!style="width: 150px" | Marker<br />
!style="width: 250px" | Full name<br />
!style="width: 250px" | Target<br />
!style="width: 450px" | Remarks<br />
!style="width: 200px" | Link<br />
|-<br />
|DAPI/Hoechst<br />
|4',6-diamidino-2-phenylindole<br />
|Nuclei<br />
|Incorporates into the DNA<br />
|[http://en.wikipedia.org/wiki/DAPI DAPI], [http://en.wikipedia.org/wiki/Hoechst_stain Hoechst]<br />
|-<br />
|PCNA<br />
|Proliferating-Cell-Nuclear-Antigen<br />
|Dividing cells<br />
|<br />
|[http://en.wikipedia.org/wiki/PCNA PCNA]<br />
|-<br />
|SOX2<br />
|SRY (sex determining region Y)-box 2<br />
|Preferentially neuronal lineage<br />
|Progenitor marker for both spinal cord and brain<br />
|[http://en.wikipedia.org/wiki/Sox2 SOX2]<br />
|-<br />
|PAX6<br />
|Paired box protein Pax-6<br />
|Brain/spinal cord<br />
|aka aniridia type II protein (AN2) or oculorhombin<br />
|[http://en.wikipedia.org/wiki/Pax6 PAX6]<br />
|-<br />
|PAX7<br />
|Paired box protein Pax-7<br />
|Satellite cells/spinal cord/brain<br />
|<br />
|[http://en.wikipedia.org/wiki/PAX7 PAX7]<br />
|-<br />
|MHC<br />
|Myosin heavy chain<br />
|Muscle fibers<br />
|<br />
|[http://en.wikipedia.org/wiki/Myosin_heavy_chain MHC]<br />
|-<br />
|MYF5<br />
|Myogenic factor 5<br />
|Myoblasts<br />
|Regulates muscle differentiation, labels cycling myoblasts<br />
|[http://en.wikipedia.org/wiki/Myf5 MYF5]<br />
|-<br />
|TUBB3<br />
|ßIII-tubulin<br />
|Axons<br />
|<br />
|[http://en.wikipedia.org/wiki/TUBB3 TUBB3]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Schwann cells<br />
|<br />
|-<br />
|PRRX1<br />
|Paired mesoderm homeobox protein 1<br />
|Connective tissue progenitors in Limb bud and Limb Blastema<br />
|<br />
|[http://en.wikipedia.org/wiki/PRRX1 PRRX1]<br />
|-<br />
|SMA<br />
|Smooth muscle actin<br />
|Blood vessels/endothelial cells<br />
|<br />
|<br />
|-<br />
|GFAP<br />
|Glial fibrillary acidic protein<br />
|Glia<br />
|<br />
|[http://en.wikipedia.org/wiki/Glial_fibrillary_acidic_protein GFAP]<br />
|-<br />
|NeuN<br />
|Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3<br />
|Differentiated nerve<br />
|<br />
|[http://en.wikipedia.org/wiki/NeuN NeuN]<br />
|-<br />
|LEU7/HNK1/B3GAT1/CD57<br />
|Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1<br />
|Schwann cells<br />
|<br />
|[http://en.wikipedia.org/wiki/B3GAT1 B3GAT1]<br />
|-<br />
|XBRA<br />
|Xbrachyury<br />
|Mesoderm during gastrula stage<br />
|<br />
|[http://en.wikipedia.org/wiki/Brachyury Brachyury]<br />
|-<br />
|MSX1<br />
|Muscle segment homeobox, Msh homeobox 1<br />
|Undifferentiated cells<br />
|Transcriptional repressor during embryogenesis<br />
|[http://en.wikipedia.org/wiki/MSX1 MSX1]<br />
|-<br />
|Trypan blue<br />
|<br />
|Dead tissues or cells<br />
|Vital stain<br />
|[http://en.wikipedia.org/wiki/Trypan_blue Trypan blue]<br />
|-<br />
|MEF2C<br />
|Myocyte-specific enhancer factor 2C<br />
|Differentiated muscle cells<br />
|aka MADS box transcription enhancer factor 2, polypeptide C<br />
|[http://en.wikipedia.org/wiki/Mef2c MEF2C]<br />
|-<br />
|PH3<br />
|Phospho-Histone H3<br />
|Mitotic cells<br />
|<br />
|<br />
|-<br />
|BrdU<br />
|Bromodeoxyuridine<br />
|Cells in S-phase<br />
|<br />
|[http://en.wikipedia.org/wiki/Brdu BrdU]<br />
|-<br />
|CASP3<br />
|Caspase 3<br />
|Dying cells<br />
|<br />
|[http://en.wikipedia.org/wiki/CASP3 CASP3]<br />
|-<br />
|GDF5<br />
|Growth/differentiation factor 5<br />
|Joints<br />
|Joint development marker<br />
|[http://en.wikipedia.org/wiki/GDF5 GDF5]<br />
|-<br />
|Survivin<br />
|<br />
|Cleavage furrow<br />
|aka baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5<br />
|[http://en.wikipedia.org/wiki/Survivin Survivin]<br />
|-<br />
|MAP2<br />
|Microtubule-associated protein 2<br />
|Neurons<br />
|<br />
|[http://en.wikipedia.org/wiki/MAP2 MAP2]<br />
|-<br />
|SOX9<br />
|SRY (sex determining region Y)-box 2<br />
|Cartillage progenitors<br />
|<br />
|[http://en.wikipedia.org/wiki/Sox9 SOX9]<br />
|-<br />
|MBP<br />
|Myelin basic protein<br />
|Myelin sheath<br />
|<br />
|[http://en.wikipedia.org/wiki/Myelin_basic_protein MBP]<br />
|}<br />
<br />
== Digital data ==<br />
Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on. <br />
Select a category from the list below in order to view or download the data.<br />
<br />
=== Fileservers ===<br />
{| class="wikitable"<br />
!style="width: 350px" | Location<br />
!style="width: 250px" | Volume size<br />
!style="width: 350px" | Description<br />
!style="width: 350px" | Access<br />
!style="width: 350px" | Location (console only)<br />
|-<br />
|//biodata/groups/<b>crtd_tanaka</b><br />
|16 TB<br />
|General purpose storage space<br />
|Entire Tanaka lab<br />
|<i>biocluster:</i> <b>/group/crtd_tanaka</b><br />
|-<br />
|//biodata/groups/<b>tanaka-ests</b><br />
|247 GB<br />
|EST sequences<br />
|Entire Tanaka lab<br />
|<i>biocluster:</i> <b>/group/tanaka-ests</b><br />
|-<br />
|//biodata/groups/<b>axolotl-transcriptome</b><br />
|500 GB<br />
|Axolotl transcriptome data<br />
|Elly, Sergej<br />
|<i>biocluster:</i> <b>/projects/axolotl-transcriptome</b><br />
|-<br />
|//biodata/groups/<b>tanaka-saori</b><br />
|878 GB<br />
|Special directory for Saori's data<br />
|Elly, Saori, Sergej<br />
|<i>biocluster:</i> <b>/group/tanaka-saori</b><br />
|}<br />
<br />
{{Tip|The path is different, when accessing the file server from the console.}}<br />
<br />
=== Literature ===<br />
<ul><br />
<li>[[Literature#Theses|Theses]]</li><br />
<li>[[Literature#Publications|Publications]]</li><br />
</ul><br />
<br />
=== Databases ===<br />
<ul><br />
<li>[http://elbrus.biochem.mpg.de/ Axolotl transcriptome] website contains the Axolotl transcriptome assemblies</li><br />
<li>[http://newtomics.mpi-bn.mpg.de/ Newt transcriptome] website contains the Newt transcriptome assembly</li><br />
<li>[http://sandberg.cmb.ki.se/redspottednewt/ Red spotted newt transcriptome] website contains the red spotted newt transcriptome assembly</li><br />
<li>[https://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi Axolotl EST] database contains several ESTs and also some additional information, e.g. the position of the well with the corresponding insert in the Blastema (BL) or Neural tube (NT) library.</li><br />
<li>[http://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi?dispatch=contig_search.cgi Short insert cDNA library]</li><br />
<li>CURRENTLY UNAVAILABLE! [http://est.age.mpg.de/ Axologle] database contains expression profiling data of Axolotl limb regeneration</li><br />
<li>CURRENTLY UNAVAILABLE! [http://est.age.mpg.de/blast/blast.html Axolotl BLAST] database contains several different BLAST-able Axolotl transciptome assemblies</li><br />
</ul><br />
<br />
=== Datasets ===<br />
<ul><br />
<li>[[Datasets#Sequences|Sequences]]<br />
<ul><br />
<li>[[Datasets#Illumina|Illumina]]</li><br />
<li>[[Datasets#Sanger|Sanger]]</li><br />
<li>[[Datasets#Roche.2F454|Roche/454]]</li><br />
</ul><br />
</li><br />
<li>[[Datasets#Microarrays|Microarrays]]<br />
</ul><br />
<br />
=== Interesting links ===<br />
<ul><br />
<li>[http://www.nytimes.com/video/2013/02/27/science/100000002087758/finding-the-visible-in-the-invisible.html Visible in the invisible]: a movie introducing a new image processing ana analysis technique developed at the MIT.</li><br />
</ul><br />
<br />
== Lab life ==<br />
<ul><br />
<li>[[Lab_photos|Lab photos]]</li><br />
<li>Further pictures can be found in <b>/biodata/groups/crtd_tanaka/LabFun/</b></li><br />
<li>The recepies for Heino's farewell present can be found under <b>/biodata/groups/crtd_tanaka/LabFun/Heino's farewell present/</b></li><br />
<li>The video of the visit of the EU commissioner can be found under <b>/biodata/groups/crtd_tanaka/LabFun/</b></li><br />
</ul></div>Sergej.nowoshilow