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== Introduction ==
== Introduction ==
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.
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.
== Axolotl ==
=== Maintainance ===
In this section you will find some general information on how to [[Axolotl#Maintaining_the_axolotls|maintain]] the animals.
=== Feeding ===
This section will give you an overview of how to [[Axolotl#Feeding|feed]] the axolotls.
=== Interesting observations ===
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.


== Lab duties ==
== Lab duties ==
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<li>[[Vacuum_pumps|Vacuum pumps]]</li>
<li>[[Vacuum_pumps|Vacuum pumps]]</li>
<li>[[Waterbath|Waterbath]]</li>
<li>[[Waterbath|Waterbath]]</li>
<li>[[RAMP Test|RAMP Test]]</li>


=== General ===
=== General ===
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</ul>
</ul>


== Antibodies ==
=== List of primary antibodies ===
{| class="wikitable sortable"
! 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
|-
| 12/101 || Developmental Studies Hybridoma Bank ||  || Mouse ||  ||  || Yuka || 4°C || 46 ug/ml Ig || 1:200 || 1:200 ||  ||  ||  ||
|-
| 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 ||
|-
| 20B4 || Developmental Studies Hybridoma Bank || 20B4 || Mouse || Monoclonal || neural crest cell (Chicken) || Yuka || 4°C ||  ||  ||  ||  ||  || http://dshb.biology.uiowa.edu/neural-crest-cells ||
|-
| 5D3 || Developmental Studies Hybridoma Bank || 5D3 || Mouse || Monoclonal || Cadherin E (Xenopus) || Yuka || 4°C ||  || 37 ug/ml IG ||  ||  ||  || http://dshb.biology.uiowa.edu/5D3 ||
|-
| 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 ||
|-
| Aggrecan || millipore || AB1031 || Rabbit ||  ||  || Lidia ||  ||  ||  || Not work, Dim || '1:500 (Lidia) ||  ||  ||
|-
| Alpha tubulin (DM1a) || MPI ||  || Mouse || Monoclonal || Chicken alpha tubulin || Takuji || -20°C ||  ||  ||  ||  || 1:1000 -  ||  ||
|-
| 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. ||
|-
| Bra ||  || AF2085 ||  ||  ||  || Yuka || -20C ||  || 0.2ug/ml in PBS ||  ||  ||  ||  ||
|-
| BrdU (Bu20a) || MPI ||  || Mouse || Monoclonal || BrdU - thymidine analog || Takuji || -20°C ||  || 1:400 -  ||  ||  ||  ||  ||
|-
| 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/ ||
|-
| Casp3 || Abcam || ab13847 || rabbit ||  ||  || Lidia || -20C ||  ||  ||  ||  ||  ||  ||
|-
| 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. ||  ||  ||  ||
|-
| col2A1 || Acris || AM00619PU-N || Mouse ||  ||  || Yuka || -20C || 0.2mg/ml in /H2O || 1:10 || 1:100-200 ||  ||  ||  ||
|-
| 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 ||
|-
| FITC || Jackson ImmunoResearch || 200-002-037 || Mouse || Monoclonal || Fluorescein isothiocyanate || Takuji || -20°C ||  || 1:400 -  ||  ||  ||  || https://www.jacksonimmuno.com/catalog/products/200-002-037 ||
|-
| FITC || invitrogen || 71-1900 || Rabbit || Polyclonal || Fluorescein isothiocyanate || Takuji || -20°C ||  || 1:400 -  ||  ||  ||  || https://www.thermofisher.com/antibody/product/FITC-Antibody-Polyclonal/71-1900 ||
|-
| GFP || Rockland || 600-401-215 || Rabbit ||  ||  || Common box || -20C ||  ||  || 1:2000-4000 ||  ||  ||  ||
|-
| GFP || MPI ||  || Goat ||  ||  || Yuka (from Jifeng) || -20C ||  || Yuka (from Jifeng) || 1:2000- ||  ||  ||  ||
|-
| GFP || MPI || 106-A20-Mix || Mouse ||  ||  || Yuka || -20C || 2.96 mg/ml in PBS ||  || 1:200, High background ||  ||  ||  ||
|-
| GFP || Abcam || ab13970 || Chick ||  ||  || Yuka (from Andrea), Lidia || -20C ||  || Andrea || High background ||  ||  ||  ||
|-
| GFP || Torrey Pines || TP401 || Rabbit ||  ||  || Yuka || -20C ||  || 1:200 (Axolotl) ||  ||  ||  ||  ||
|-
| GFP || Invitrogen || A11120 || Mouse || monoclonal ||  || Common box || -20C || 1 mg/ml in PBS ||  ||  ||  ||  ||  ||
|-
| Hoxa11 || Tanaka Lab (Kathleen) ||  || Rabbit || Polyclonal  || Axolotl || Yuka_Aliquot from Kathleen || -20C || 0.95 mg/ml ||  || FF+2%MEMFA.Dent's ||  ||  ||  ||
|-
| Hoxa11 || Tanaka Lab (Kathleen) ||  || Mouse || Monoclonal || Axolotl || Yuka_Aliquot from Kathleen || -20C || 4.22 mg/ml ||  ||  ||  ||  ||  ||
|-
| Hoxa13 || Tanaka Lab (Kathleen) ||  || Rabbit || Polyclonal  || Axolotl || Yuka_Aliquot from Kathleen || -20C || 0.15 mg/ml || 01:50 || 1:50, FreshFrozen+Unfixed ||  ||  ||  ||
|-
| Hoxa9 || Tanaka Lab (Kathleen) ||  || Rabbit || Polyclonal  || Axolotl || Yuka_Aliquot from Kathleen || -20C || 1.35 mg/ml ||  || FF+Methanol+ph9AR ||  ||  ||  ||
|-
| Laminin || Sigma || L9393 || Rabbit ||  ||  || Yuka (from Jifeng) || -20C ||  ||  || 1:200, 500_1:25 (paraffin), 1:100 (Slack) ||  ||  ||  ||
|-
| MBP || Genetex || GTX76114 || Rat || Monoclonal || Myelin basic protein (Bovine) || Common box || -20C ||  || 1:200 || 1:200 ||  ||  ||  ||
|-
| 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) ||  ||  ||  ||
|-
| 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 ||
|-
| MHC || Developmental Studies Hybridoma Bank || A4.1025 || Mouse ||  ||  || Yuka || 4°C ||  || 1:200 || 1:200 ||  ||  ||  ||
|-
| Osterix || abcam || ab22552 || rabbbit ||  ||  || Lidia ||  ||  ||  ||  ||  ||  ||  ||
|-
| Pax7 || MPI ||  || Mouse ||  ||  || Yuka || -20C ||  || 1:100, 200, 400  ||  ||  ||  ||  ||
|-
| PCNA || Santa Cruz || sc-56 AF64 || Alexa Fluor -647  ||  ||  || Lidia ||  ||  ||  || 1:500 ||  ||  ||  ||
|-
| 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 ||
|-
| Perilipin || R and D || P1873 || rabbit ||  ||  || Lidia ||  ||  ||  ||  ||  ||  ||  ||
|-
| 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 ||
|-
| PPARgamma || Cell signalling || 81B8 || rabbit ||  ||  || Lidia ||  ||  ||  ||  ||  ||  ||  ||
|-
| Prrx1 || Tanaka Lab (Prayag) ||  || Rabbit || Polyclonal || Axolotl N-terminus (1-101 aa) || Lidia, Yuka_Aliquot from Prayag || -20C ||  || 1:200 || 1:200 ||  ||  ||  ||
|-
| 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 ||
|-
| Rhodamine || Molecular Probes || A-6397 || Rabbit || Polyclonal || Tetramethylrhodamine || Takuji || -20°C ||  || 1:400 -  ||  ||  ||  ||  ||
|-
| Scleraxis || Santa Cruz || D-14, sc-87425 || Goat || Polyclonal Goat IgG ||  || Yuka || 4°C ||  ||  ||  ||  ||  ||  ||
|-
| Shh (H-160) || Santa Cruz || SC-9024 || Rabbit || Polyclonal ||  || Yuka || 4°C ||  || 200 ug/ml ||  ||  ||  ||  ||
|-
| 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 ||
|-
| 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 ||
|-
| Zo-1 || Invitrogen || 33-9100 || Mouse ||  ||  || Yuka (from Akira) || -20C ||  ||  ||  ||  ||  ||  ||
|-
| GADD45B || SIGMA || SAB2108614-100 || Rabbit || Polyclonal || QIHFT LIQSF CCDND INIVR VSGMQ RLAQL LGEPA ETQGT TEARD LHCLL || Marco || -20C || 0.5-1 mg/ml ||  ||  ||  ||  ||  ||
|-
| 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 ||  ||  ||  ||  ||  ||
|-
| 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 ||  ||  ||  ||  ||  ||
|-
| Tet3 || Diagenode || C15410311 || Rabbit || Polyclonal ||  || Marco || -20C || 1 ug/uL ||  ||  ||  ||  ||  ||
|-
| Tet2 || Diagenode || C15410255 || Rabbit || Polyclonal ||  || Marco || -20C || 1 ug/uL ||  ||  ||  ||  ||  ||
|-
| 5-mC || Diagenode || C15200081-100 || Mouse || Monoclonal ||  || Marco || -20C || 1.36 ug/ uL ||  ||  ||  ||  ||  ||
|-
| 5-hmC || Diagenode || C15410205-20 || Rabbit || Polyclonal ||  || Marco || -20C || 3.5 ug/uL ||  ||  ||  ||  ||  ||
|-
| IgG (Rabbit) || Diagenode || C15410206 || Rabbit || Polyclonal ||  || Marco || -20C || 1 ug/uL ||  ||  ||  ||  ||  ||
|-
| IgG (Rabbit) || CST || 2729S || Rabbit || Polyclonal ||  || Akane || -20C || 1-5ug/uL ||  ||  ||  ||  || https://media.cellsignal.com/pdf/2729.pdf#_ga=2.22008443.1736501072.1510317731-1668154722.1509367929 ||
|-
|anti-H3 (Rabbit) || Abcam || ab1791 || Rabbit || Polyclonal ||  || Akane || -20C || 1 ug/uL || 1:5000(WB), 1:1000 (CHiP), 1:1000 (Tissue IF)||  ||  ||  || Synthetic peptide corresponding to Human Histone H3 aa 100 to the C-terminus conjugated to Keyhole Limpet Haemocyanin (KLH).http://www.abcam.co.jp/histone-h3-antibody-nuclear-loading-control-and-chip-grade-ab1791.html ||
|-
|anti-H3K4me3 (Rabbit) || Abcam || ab8580 || Rabbit || Polyclonal ||  || Akane || -20C || 1 ug/uL || 1:1000 (WB), 1:250 (CHiP), 1:500 (Tissue IF)||  ||  ||  || Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.(http://www.abcam.co.jp/histone-h3-tri-methyl-k4-antibody-chip-grade-ab8580.html)||
|-
|anti-anti-H3K4me1 (Rabbit) ||　Abcam || ab8895||　Rabbit || Polyclonal ||  || Akane || -20C || 0,2 ug/uL || 1:500 (WB), 1:250 (CHiP), 1:500 (Tissue IF)||  ||  ||  || Synthetic peptide within Human Histone H3 aa 1-100 (mono methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH). http://www.abcam.co.jp/histone-h3-mono-methyl-k4-antibody-chip-grade-ab8895.html||
|-
|anti-H3K9me3 (Rabbit) || Abcam || ab8898|| Rabbit || Polyclonal ||  || Akane || -20C || 1 ug/uL || 1:1000 (WB), 1:500 (Tissue IF)||  ||  ||  || Synthetic peptide within Human Histone H3 aa 1-100 (N terminal) (tri methyl K9) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
(Peptide available as ab1773) http://www.abcam.co.jp/histone-h3-tri-methyl-k9-antibody-chip-grade-ab8898.html||
|-
|anti-H3K27ac (Rabbit) || Abcam || ab4729|| Rabbit || Polyclonal ||  || Akane || -20C || 1 ug/uL || 1:1000 (WB), 1:500 (Tissue IF)||  ||  ||  || Synthetic peptide corresponding to Human Histone H3 aa 1-100 (acetyl K27) conjugated to Keyhole Limpet Haemocyanin (KLH). http://www.abcam.co.jp/histone-h3-acetyl-k27-antibody-chip-grade-ab4729.html||
|-
|anti-H3K27me3 (Rabbit) || millipore || 07-449 || Rabbit || Polyclonal ||  || Akane || -20C || 1 ug/uL || 1:1000 (WB), 1:500 (Tissue IF)||  ||  ||  || KLH-conjugated, synthetic 2X-branched peptide containing the sequence …AR(me3K)SAP… in which me3K corresponds to trimethyl-lysine at residue 27 of human histone H3. http://www.merckmillipore.com/AT/de/product/Anti-trimethyl-Histone-H3-Lys27-Antibody,MM_NF-07-449?ReferrerURL=https%3A%2F%2Fwww.google.at%2F&bd=1||
|-
|anti-Ezh2 mAB (mouse) || CST || CST3147  || mouse || mono clonal ||  || Akane || -20C || 1 ug/uL || WB, IF, ChIP DIDNT WORK ||  ||  ||  || https://www.cellsignal.com/products/primary-antibodies/ezh2-ac22-mouse-mab/3147 ||
|-
|Pol II antibody  (mouse) || CST || C15100055-100  || mouse|| mono clonal ||  || Akane || -20C || 1 ug/uL ||  ||  ||  ||  || mouse against the B1 subunit of RNA polymerase II (polymerase (RNA) II (DNA directed) polypeptide A) of wheat germ. Interacts with the highly conserved C-terminal domain of the protein containing the YSPTSPS repeat. https://www.diagenode.com/files/products/antibodies/Datasheet_PoII_C15100055.pdf||
|-
| IgG (mouse) || CST ||  || Rabit || Mono clonal || || Akane || -20C || 1 ug/uL || WB, IF, ChIP DIDNT WORK ||  ||  ||  ||  ||
|-
| CTCF (D31H2) XP®  || CST ||  CST-3418S|| Rabbit || Mono clonal ||  || Akane || -20C || 1 ug/uL || WB, IF, ChIP DIDNT WORK ||  ||  ||  ||  ||
|}


== Protocols ==
== Protocols ==
Line 44: Line 172:
<li>[[Adhesive_cell_culture|Adhesive cell culture]]</li>
<li>[[Adhesive_cell_culture|Adhesive cell culture]]</li>
<li>[[Baculovirus_titration|Baculovirus titration]]</li>
<li>[[Baculovirus_titration|Baculovirus titration]]</li>
<li>[[Cardiomyocyte_Preparation|Cardiomyocyte preparation]]</li>
<li>[[Electroporation_of_nec|Electroporation of neural epithelia cells (axolotl spinal cord)]]</li>
<li>[[Electroporation_of_nec|Electroporation of neural epithelia cells (axolotl spinal cord)]]</li>
<li>[[Myoblasts_electroporation|Myoblasts electroporation]]</li>
<li>[[Myoblasts_electroporation|Myoblasts electroporation]]</li>
<li>[[Neurosphere_culture|Neurosphere culture]]</li>
<li>[[Neurosphere_culture|Neurosphere culture]]</li>
<li>[[Preparation_of_Cardiomyocytes|Preparation of cardiomyocytes]]</li>
<li>[[Thawing_293FT_cells|Thawing 293FT cells]]</li>
<li>[[Thawing_293FT_cells|Thawing 293FT cells]]</li>
<li>[[G418_medium_preparation|G418 medium preparation]]</li>
<li>[[Fibronectin_Preparation|Fibronectin preparation]]</li>
<li>[[Gelatin Preparation|Gelatin preparation]]</li>
<li>[[Insulin_Preparation|Insulin preparation]]</li>
<li>[[Insulin_Preparation|Insulin preparation]]</li>
<li>[[Gelatin Embedding|Gelatin Embedding]]</li>


===== A1 cells =====
===== A1 cells =====
<li>[[A1_cell_passaging|A1 cell passaging]]</li>
 
<li>[[Thawing_frozen_A1_cells|Thawing frozen A1 cells]]</li>
<li>[[Freezing_of_A1_cells|Freezing of A1 cells]]</li>
<li>[[Passaging_A1_cell|A1 cell passaging]]</li>
<li>[[A1_cell_passaging_myotube|A1 cell passaging for myotube preparation]]</li>
<li>[[Modified_myotube_prep|Modified myotube prep]]</li>
<li>[[A1_cell_transfection_with_FuGene|A1 cell transfection with FuGene]]</li>
<li>[[Electroporation_of_A1_cells|Electroporation of A1 cells]]</li>
<li>[[BrdU_labelling_of_A1_cells_in_96-well_plate|BrdU labelling of A1 cells in 96-well plate]]</li>
<li>[[BrdU_labelling_of_A1_cells_in_96-well_plate|BrdU labelling of A1 cells in 96-well plate]]</li>
<li>[[BrdU_and_myosin_staining|BrdU and myosin staining]]</li>
<li>[[Preparation_of_Low_Serum_for_A1_cells|Preparation of Low Serum for A1 cells]]</li>
<li>[[Preparation_of_High_Serum_for_A1_cells|Preparation of High Serum for A1 cells]]</li>
<li>[[Preparation_of_Freezing_media_for_A1_cells|Preparation of Freezing media for A1 cells]]</li>


===== Blastema cells =====
===== Blastema cells =====
<li>[[Blastema_cells|Blastema cells]]</li>
<li>[[Blastema_cells|Blastema cells]]</li>
<li>[[Electroporation_of_blastema_cells|Electroporation of blastema cells]]</li>
<li>[[Electroporation_of_blastema_cells|Electroporation of blastema cells]]</li>
===== C2C12 cells =====
<li>[[Thawing_frozen_C2C12_cells|Thawing frozen C2C12 cells]]</li>
<li>[[Freezing_of_C2C12_cells|Freezing of C2C12 cells]]</li>
<li>[[Passaging_C2C12_cells|C2C12 cell passaging]]</li>
<li>[[Myotube_preparation_of_C2C12_cells|Myotube preparation of C2C12 cells]]</li>
<li>[[C2C12_cell_transfection_with_FuGene|C2C12 cell transfection with FuGene]]</li>
<li>[[Cloning_of_C2C12_cells|Cloning of C2C12 cells]]</li>
<li>[[Electroporation_of_C2C12_cells|Electroporation of C2C12 cells]]</li>
<li>[[Preparation_of_Low_Serum_for_C2C12_cells|Preparation of Low Serum for C2C12 cells]]</li>
<li>[[Preparation_of_High_Serum_for_C2C12_cells|Preparation of High Serum for C2C12 cells]]</li>
<li>[[Preparation_of_Freezing_media_for_C2C12_cells|Preparation of Freezing media for C2C12 cells]]</li>


===== Embryonic stem cells =====
===== Embryonic stem cells =====
Line 71: Line 229:
<li>[[TSA_Staining_on_axolotl-tissue|Tyramide signal amplification (TSA) staining on axolotl tissue]]</li>
<li>[[TSA_Staining_on_axolotl-tissue|Tyramide signal amplification (TSA) staining on axolotl tissue]]</li>


=== Antibody ===
===== In situ Hybridization =====
===== Monoclonal Antibodies: =====
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Anja)]]</li>
<li>[[ISH_on_axolotl_tissue_sections|ISH on axolotl tissue sections (Akira)]]</li>
<li>[[Whole_mount_ISH_on_axolotl_tissue|Whole mount ISH on axolotl tissue]]</li>
 
=== Antibodies ===
 
===== Monoclonal Antibodies =====
<li>[[Purifying IgG1 from Hybridoma Supernatant]]</li>
<li>[[Purifying IgG1 from Hybridoma Supernatant]]</li>
<li>[[Labelling myosin antibody]]</li>
<li>[[Labelling myosin antibody]]</li>
<li>[[Labelling Pax6 with Dig-NHS]]</li>
<li>[[Labelling Pax6 with Dig-NHS]]</li>
<li>[[Anti-BrDU Antibody labeling]]</li>


===== Polyclonal Antibodies: =====
===== Polyclonal Antibodies =====
<li>[[Addresses for making polyclonal antibodies]]</li>
<li>[[Address for making polyclonal antibodies]]</li>
<li>[[Preparation of antigen for antibody production]]</li>
<li>[[Letter to Froppier (French)]]</li>
<li>[[Letter to Froppier (French)]]</li>
<li>[[labeled AB staining]]</li>
<li>[[labeled AB staining]]</li>
<li>[[Antibody staining]]</li>
<li>[[Antibody staining]]</li>
<li>[[Antibody purification]]</li>
<li>[[Antibody purification Sox2]]</li>
===== ELISA =====
<li>[[ELISA]]</li>


=== Molecular biology ===
=== Molecular biology ===
<li>[[Metamorphosis_protocol|Metamorphosis protocol (Sherry)]]</li>
<li>[[Metamorphosis_protocol|Metamorphosis protocol]]</li>
<li>[[Digestion_DNA|DNA Digestion (Plasmid)]]</li>
<li>[[Digestion_DNA|Plasmid digestion]]</li>
<li>[[RNA_Extraction_from_axolotl_tissue|RNA Extraction (axolotl tissue)]]</li>
<li>[[RNA_Extraction_from_axolotl_tissue|RNA Extraction (axolotl tissue)]]</li>
<li>[[RNA_formaldehyde_gels|RNA formaldehyde gels]]</li>
<li>[[RNA_formaldehyde_gels|RNA formaldehyde gels]]</li>
 
<li>[[Amputation_andRNA_Isolation|Amputation and RNA Isolation]]</li>
== Lab business ==
<li>[[Linker Insertion|Linker Insertion]]</li>
=== Ordering database ===
<li>[[gRNA Production|gRNA Production]]</li>
Use the following [http://ordering-db.biotec.tu-dresden.de/ordering/login.gsp ordering database] to place your orders.
<li>[[Cocktail Protocol for Pax7|Cocktail Protocol for Pax7]]</li>
 
=== Booking calendars ===
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.
<ul>
<li>
    Fluorescence microscopes
    <ul>
      <li>[http://www.my.calendars.net/olympusszx16 Olympus SZX 16]</li>
      <li>[http://www.my.calendars.net/new_olympus/ New Olympus SZX 16]</li>
      <li>[http://www.my.calendars.net/Olympus_OVK Olympus OVK]</li>
      <li>[http://www.my.calendars.net/axio_observer/ Axio Observer]</li>
    </ul>
</li>
<li>
    Microinjectors and electroporators and dissecting microscope
    <ul>
      <li>Room 0.232
          <ul>         
              <li>[http://www.my.calendars.net/electroporator Nepa Gene]</li>
              <li>[http://www.my.calendars.net/electroporator2 ECM 830]</li>
              <li>[http://www.my.calendars.net/stereoscope2/d01/11/2012 Olympus dissecting microscope]</li>
          </ul>
      </li>
      <li>Lab
          <ul>
              <li>[http://www.my.calendars.net/OlympusLab Olympus (next to the axolotls)]</li>
              <li>[http://www.my.calendars.net/OlympusLab2 Olympus (next to the Microtome)]</li>
        </ul>
      </li>
    </ul>
</li>
<li>
    Confocal microscopes
    <ul>
      <li>[http://www.my.calendars.net/leica_lsm Leica LSM]</li>
    </ul>
</li>
<li>
    Cryostats
    <ul>
      <li>[http://www.my.calendars.net/cryostat1 Cryostat 1]</li>
      <li>[http://www.my.calendars.net/cryostat2 Cryostat 2]</li>
    </ul>
</li>
<li>
  Microtome
  <ul>
      <li>[http://www.my.calendars.net/microtome Microtome]</li>
  </ul>
</li>
<li>
  Paraffin embedding station
  <ul>
      <li>[http://www.my.calendars.net/wax_station Paraffin embedding station]</li>
  </ul>
</li>
</ul>


== Genetics ==
== Genetics ==
Line 325: Line 437:
Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on.  
Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on.  
Select a category from the list below in order to view or download the data.
Select a category from the list below in order to view or download the data.
=== Fileservers ===
{| class="wikitable"
!style="width: 350px" | Location
!style="width: 250px" | Volume size
!style="width: 350px" | Description
!style="width: 350px" | Access
!style="width: 350px" | Location (console only)
|-
|//biodata/groups/<b>crtd_tanaka</b>
|16 TB
|General purpose storage space
|Entire Tanaka lab
|<i>biocluster:</i> <b>/group/crtd_tanaka</b>
|-
|//biodata/groups/<b>tanaka-ests</b>
|247 GB
|EST sequences
|Entire Tanaka lab
|<i>biocluster:</i> <b>/group/tanaka-ests</b>
|-
|//biodata/groups/<b>axolotl-transcriptome</b>
|500 GB
|Axolotl transcriptome data
|Elly, Sergej
|<i>biocluster:</i> <b>/projects/axolotl-transcriptome</b>
|-
|//biodata/groups/<b>tanaka-saori</b>
|878 GB
|Special directory for Saori's data
|Elly, Saori, Sergej
|<i>biocluster:</i> <b>/group/tanaka-saori</b>
|}
{{Tip|The path is different, when accessing the file server from the console.}}


=== Literature ===
=== Literature ===
Line 369: Line 446:
=== Databases ===
=== Databases ===
<ul>
<ul>
     <li>[http://elbrus.biochem.mpg.de/ Axolotl transcriptome] website contains the Axolotl transcriptome assemblies</li>
     <li>[https://axolotl-omics.org Axolotl transcriptome] website contains the Axolotl transcriptome assemblies</li>
    <li>[https://genome.axolotl-omics.org Axolotl genome] website contains the Axolotl genome assemblies</li>
     <li>[http://newtomics.mpi-bn.mpg.de/ Newt transcriptome] website contains the Newt transcriptome assembly</li>
     <li>[http://newtomics.mpi-bn.mpg.de/ Newt transcriptome] website contains the Newt transcriptome assembly</li>
     <li>[http://sandberg.cmb.ki.se/redspottednewt/ Red spotted newt transcriptome] website contains the red spotted newt transcriptome assembly</li>
     <li>[http://sandberg.cmb.ki.se/redspottednewt/ Red spotted newt transcriptome] website contains the red spotted newt transcriptome assembly</li>
     <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>
     <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>
     <li>[http://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi?dispatch=contig_search.cgi Short insert cDNA library]</li>
     <li>[http://python-srv1.mpi-cbg.de/axolotl/cgi-bin/login.cgi?dispatch=contig_search.cgi Short insert cDNA library]</li>
     <li>CURRENTLY UNAVAILABLE! [http://est.age.mpg.de/ Axologle] database contains expression profiling data of Axolotl limb regeneration</li>
     <li>[https://axologl.axolotl-omics.org Axologle] database contains expression profiling data of Axolotl limb regeneration</li>
     <li>CURRENTLY UNAVAILABLE! [http://est.age.mpg.de/blast/blast.html Axolotl BLAST] database contains several different BLAST-able Axolotl transciptome assemblies</li>
     <li>[https://axologl.axolotl-omics.org/blast/blast.html Axolotl BLAST] database contains several different BLAST-able Axolotl transciptome assemblies</li>
</ul>
</ul>


Line 393: Line 471:
<ul>
<ul>
   <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>
   <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>
</ul>
== Lab life ==
<ul>
  <li>[[Lab_photos|Lab photos]]</li>
  <li>Further pictures can be found in <b>/biodata/groups/crtd_tanaka/LabFun/</b></li>
  <li>The recepies for Heino's farewell present can be found under <b>/biodata/groups/crtd_tanaka/LabFun/Heino's farewell present/</b></li>
  <li>The video of the visit of the EU commissioner can be found under <b>/biodata/groups/crtd_tanaka/LabFun/</b></li>
</ul>
</ul>

Latest revision as of 15:37, 19 July 2022

Introduction

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.

Lab duties

Antibodies

List of primary antibodies

Name Supplier Cat. No Host Mono/Poly Antigen Contact person Storage temp Stock concentration Conditions for axolotl Conditions for Xenopus Conditions for mouse Conditions for cell culture Reference
12/101 Developmental Studies Hybridoma Bank Mouse Yuka 4°C 46 ug/ml Ig 1:200 1:200
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
20B4 Developmental Studies Hybridoma Bank 20B4 Mouse Monoclonal neural crest cell (Chicken) Yuka 4°C http://dshb.biology.uiowa.edu/neural-crest-cells
5D3 Developmental Studies Hybridoma Bank 5D3 Mouse Monoclonal Cadherin E (Xenopus) Yuka 4°C 37 ug/ml IG http://dshb.biology.uiowa.edu/5D3
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
Aggrecan millipore AB1031 Rabbit Lidia Not work, Dim '1:500 (Lidia)
Alpha tubulin (DM1a) MPI Mouse Monoclonal Chicken alpha tubulin Takuji -20°C 1:1000 -
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.
Bra AF2085 Yuka -20C 0.2ug/ml in PBS
BrdU (Bu20a) MPI Mouse Monoclonal BrdU - thymidine analog Takuji -20°C 1:400 -
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/
Casp3 Abcam ab13847 rabbit Lidia -20C
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.
col2A1 Acris AM00619PU-N Mouse Yuka -20C 0.2mg/ml in /H2O 1:10 1:100-200
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
FITC Jackson ImmunoResearch 200-002-037 Mouse Monoclonal Fluorescein isothiocyanate Takuji -20°C 1:400 - https://www.jacksonimmuno.com/catalog/products/200-002-037
FITC invitrogen 71-1900 Rabbit Polyclonal Fluorescein isothiocyanate Takuji -20°C 1:400 - https://www.thermofisher.com/antibody/product/FITC-Antibody-Polyclonal/71-1900
GFP Rockland 600-401-215 Rabbit Common box -20C 1:2000-4000
GFP MPI Goat Yuka (from Jifeng) -20C Yuka (from Jifeng) 1:2000-
GFP MPI 106-A20-Mix Mouse Yuka -20C 2.96 mg/ml in PBS 1:200, High background
GFP Abcam ab13970 Chick Yuka (from Andrea), Lidia -20C Andrea High background
GFP Torrey Pines TP401 Rabbit Yuka -20C 1:200 (Axolotl)
GFP Invitrogen A11120 Mouse monoclonal Common box -20C 1 mg/ml in PBS
Hoxa11 Tanaka Lab (Kathleen) Rabbit Polyclonal Axolotl Yuka_Aliquot from Kathleen -20C 0.95 mg/ml FF+2%MEMFA.Dent's
Hoxa11 Tanaka Lab (Kathleen) Mouse Monoclonal Axolotl Yuka_Aliquot from Kathleen -20C 4.22 mg/ml
Hoxa13 Tanaka Lab (Kathleen) Rabbit Polyclonal Axolotl Yuka_Aliquot from Kathleen -20C 0.15 mg/ml 01:50 1:50, FreshFrozen+Unfixed
Hoxa9 Tanaka Lab (Kathleen) Rabbit Polyclonal Axolotl Yuka_Aliquot from Kathleen -20C 1.35 mg/ml FF+Methanol+ph9AR
Laminin Sigma L9393 Rabbit Yuka (from Jifeng) -20C 1:200, 500_1:25 (paraffin), 1:100 (Slack)
MBP Genetex GTX76114 Rat Monoclonal Myelin basic protein (Bovine) Common box -20C 1:200 1:200
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)
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
MHC Developmental Studies Hybridoma Bank A4.1025 Mouse Yuka 4°C 1:200 1:200
Osterix abcam ab22552 rabbbit Lidia
Pax7 MPI Mouse Yuka -20C 1:100, 200, 400
PCNA Santa Cruz sc-56 AF64 Alexa Fluor -647 Lidia 1:500
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
Perilipin R and D P1873 rabbit Lidia
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
PPARgamma Cell signalling 81B8 rabbit Lidia
Prrx1 Tanaka Lab (Prayag) Rabbit Polyclonal Axolotl N-terminus (1-101 aa) Lidia, Yuka_Aliquot from Prayag -20C 1:200 1:200
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
Rhodamine Molecular Probes A-6397 Rabbit Polyclonal Tetramethylrhodamine Takuji -20°C 1:400 -
Scleraxis Santa Cruz D-14, sc-87425 Goat Polyclonal Goat IgG Yuka 4°C
Shh (H-160) Santa Cruz SC-9024 Rabbit Polyclonal Yuka 4°C 200 ug/ml
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
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
Zo-1 Invitrogen 33-9100 Mouse Yuka (from Akira) -20C
GADD45B SIGMA SAB2108614-100 Rabbit Polyclonal QIHFT LIQSF CCDND INIVR VSGMQ RLAQL LGEPA ETQGT TEARD LHCLL Marco -20C 0.5-1 mg/ml
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
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
Tet3 Diagenode C15410311 Rabbit Polyclonal Marco -20C 1 ug/uL
Tet2 Diagenode C15410255 Rabbit Polyclonal Marco -20C 1 ug/uL
5-mC Diagenode C15200081-100 Mouse Monoclonal Marco -20C 1.36 ug/ uL
5-hmC Diagenode C15410205-20 Rabbit Polyclonal Marco -20C 3.5 ug/uL
IgG (Rabbit) Diagenode C15410206 Rabbit Polyclonal Marco -20C 1 ug/uL
IgG (Rabbit) CST 2729S Rabbit Polyclonal Akane -20C 1-5ug/uL https://media.cellsignal.com/pdf/2729.pdf#_ga=2.22008443.1736501072.1510317731-1668154722.1509367929
anti-H3 (Rabbit) Abcam ab1791 Rabbit Polyclonal Akane -20C 1 ug/uL 1:5000(WB), 1:1000 (CHiP), 1:1000 (Tissue IF) Synthetic peptide corresponding to Human Histone H3 aa 100 to the C-terminus conjugated to Keyhole Limpet Haemocyanin (KLH).http://www.abcam.co.jp/histone-h3-antibody-nuclear-loading-control-and-chip-grade-ab1791.html
anti-H3K4me3 (Rabbit) Abcam ab8580 Rabbit Polyclonal Akane -20C 1 ug/uL 1:1000 (WB), 1:250 (CHiP), 1:500 (Tissue IF) Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.(http://www.abcam.co.jp/histone-h3-tri-methyl-k4-antibody-chip-grade-ab8580.html)
anti-anti-H3K4me1 (Rabbit) 　Abcam ab8895 　Rabbit Polyclonal Akane -20C 0,2 ug/uL 1:500 (WB), 1:250 (CHiP), 1:500 (Tissue IF) Synthetic peptide within Human Histone H3 aa 1-100 (mono methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH). http://www.abcam.co.jp/histone-h3-mono-methyl-k4-antibody-chip-grade-ab8895.html
anti-H3K9me3 (Rabbit) Abcam ab8898 Rabbit Polyclonal Akane -20C 1 ug/uL 1:1000 (WB), 1:500 (Tissue IF) Synthetic peptide within Human Histone H3 aa 1-100 (N terminal) (tri methyl K9) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.

(Peptide available as ab1773) http://www.abcam.co.jp/histone-h3-tri-methyl-k9-antibody-chip-grade-ab8898.html%7C%7C

anti-H3K27ac (Rabbit) Abcam ab4729 Rabbit Polyclonal Akane -20C 1 ug/uL 1:1000 (WB), 1:500 (Tissue IF) Synthetic peptide corresponding to Human Histone H3 aa 1-100 (acetyl K27) conjugated to Keyhole Limpet Haemocyanin (KLH). http://www.abcam.co.jp/histone-h3-acetyl-k27-antibody-chip-grade-ab4729.html
anti-H3K27me3 (Rabbit) millipore 07-449 Rabbit Polyclonal Akane -20C 1 ug/uL 1:1000 (WB), 1:500 (Tissue IF) KLH-conjugated, synthetic 2X-branched peptide containing the sequence …AR(me3K)SAP… in which me3K corresponds to trimethyl-lysine at residue 27 of human histone H3. http://www.merckmillipore.com/AT/de/product/Anti-trimethyl-Histone-H3-Lys27-Antibody,MM_NF-07-449?ReferrerURL=https%3A%2F%2Fwww.google.at%2F&bd=1
anti-Ezh2 mAB (mouse) CST CST3147 mouse mono clonal Akane -20C 1 ug/uL WB, IF, ChIP DIDNT WORK https://www.cellsignal.com/products/primary-antibodies/ezh2-ac22-mouse-mab/3147
Pol II antibody (mouse) CST C15100055-100 mouse mono clonal Akane -20C 1 ug/uL mouse against the B1 subunit of RNA polymerase II (polymerase (RNA) II (DNA directed) polypeptide A) of wheat germ. Interacts with the highly conserved C-terminal domain of the protein containing the YSPTSPS repeat. https://www.diagenode.com/files/products/antibodies/Datasheet_PoII_C15100055.pdf
IgG (mouse) CST Rabit Mono clonal Akane -20C 1 ug/uL WB, IF, ChIP DIDNT WORK
CTCF (D31H2) XP® CST CST-3418S Rabbit Mono clonal Akane -20C 1 ug/uL WB, IF, ChIP DIDNT WORK

Protocols

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