Overview
Revision as of 11:21, 19 May 2020 by Sergej.nowoshilow (talk | contribs) (→List of primary antibodies)
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.
Axolotl
Maintainance
In this section you will find some general information on how to maintain the animals.
Mating
In this section you will find the information on how to mate axolotls.
Feeding
This section will give you an overview of how to feed the axolotls.
Axolotl in Vitro Fertilization
This section will give you an overview of how to In vitro fertilize the axolotls.
Interesting observations
In the following section you can find some interesting observations concerning the axolotl, its behavior, caveats of the transgenics and many more.
Genotyping (galaxy)
This section section will give you an overview of how to genotype the axolotls.
Lab duties
- Antibiotics
- Benzocaine
- Fibronectin
- Gelatine
- Liquid nitrogen
- Primary antibodies
- Secondary antibodies
- Serum heat inactivation
- Autoclave
- Cryostat
- Dissecting microscopes
- Leica confocal microscope
- Vacuum pumps
- Waterbath
- RAMP Test
- Ordering common stuff
- Heat-shock competent cells
Chemicals
Hardware
General
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®ion=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 |
Protocols
- Adhesive cell culture
- Baculovirus titration
- Cardiomyocyte preparation
- Electroporation of neural epithelia cells (axolotl spinal cord)
- Myoblasts electroporation
- Neurosphere culture
- Thawing 293FT cells
- G418 medium preparation
- Fibronectin preparation
- Gelatin preparation
- Insulin preparation
- Insulin preparation
- Gelatin Embedding
- Thawing frozen A1 cells
- Freezing of A1 cells
- A1 cell passaging
- A1 cell passaging for myotube preparation
- Modified myotube prep
- A1 cell transfection with FuGene
- Electroporation of A1 cells
- BrdU labelling of A1 cells in 96-well plate
- BrdU and myosin staining
- Preparation of Low Serum for A1 cells
- Preparation of High Serum for A1 cells
- Preparation of Freezing media for A1 cells
- Blastema cells
- Electroporation of blastema cells
- Thawing frozen C2C12 cells
- Freezing of C2C12 cells
- C2C12 cell passaging
- Myotube preparation of C2C12 cells
- C2C12 cell transfection with FuGene
- Cloning of C2C12 cells
- Electroporation of C2C12 cells
- Preparation of Low Serum for C2C12 cells
- Preparation of High Serum for C2C12 cells
- Preparation of Freezing media for C2C12 cells
- mESC culture/Cyst formation
- Thawing (XB10) hybridoma cells
- Hybridoma cells in SFX media
- Hybridoma cell clones
- Alcian Blue Staining
- Double Immunostaining via antigen retrieval (Sherry)
- Tyramide signal amplification (TSA) staining on axolotl tissue
- ISH on axolotl tissue sections (Anja)
- ISH on axolotl tissue sections (Akira)
- Whole mount ISH on axolotl tissue
- Purifying IgG1 from Hybridoma Supernatant
- Labelling myosin antibody
- Labelling Pax6 with Dig-NHS
- Anti-BrDU Antibody labeling
- Address for making polyclonal antibodies
- Letter to Froppier (French)
- labeled AB staining
- Antibody staining
- Antibody purification
- Antibody purification Sox2
- ELISA
- Metamorphosis protocol
- Plasmid digestion
- RNA Extraction (axolotl tissue)
- RNA formaldehyde gels
- Amputation and RNA Isolation
- Linker Insertion
- gRNA Production
- Cocktail Protocol for Pax7
- Axolotl transcriptome website contains the Axolotl transcriptome assemblies
- Axolotl genome website contains the Axolotl genome assemblies
- Newt transcriptome website contains the Newt transcriptome assembly
- Red spotted newt transcriptome website contains the red spotted newt transcriptome assembly
- 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.
- Short insert cDNA library
- Axologle database contains expression profiling data of Axolotl limb regeneration
- Axolotl BLAST database contains several different BLAST-able Axolotl transciptome assemblies
- Visible in the invisible: a movie introducing a new image processing ana analysis technique developed at the MIT.
Cell culture
A1 cells
Blastema cells
C2C12 cells
Embryonic stem cells
Hybridoma cells
Histology
In situ Hybridization
Antibodies
Monoclonal Antibodies
Polyclonal Antibodies
ELISA
Molecular biology
Genetics
Transgenic lines
Markers
| Marker | Full name | Target | Remarks | Link |
|---|---|---|---|---|
| DAPI/Hoechst | 4',6-diamidino-2-phenylindole | Nuclei | Incorporates into the DNA | DAPI, Hoechst |
| PCNA | Proliferating-Cell-Nuclear-Antigen | Dividing cells | PCNA | |
| SOX2 | SRY (sex determining region Y)-box 2 | Preferentially neuronal lineage | Progenitor marker for both spinal cord and brain | SOX2 |
| PAX6 | Paired box protein Pax-6 | Brain/spinal cord | aka aniridia type II protein (AN2) or oculorhombin | PAX6 |
| PAX7 | Paired box protein Pax-7 | Satellite cells/spinal cord/brain | PAX7 | |
| MHC | Myosin heavy chain | Muscle fibers | MHC | |
| MYF5 | Myogenic factor 5 | Myoblasts | Regulates muscle differentiation, labels cycling myoblasts | MYF5 |
| TUBB3 | ßIII-tubulin | Axons | TUBB3 | |
| MBP | Myelin basic protein | Schwann cells | ||
| PRRX1 | Paired mesoderm homeobox protein 1 | Connective tissue progenitors in Limb bud and Limb Blastema | PRRX1 | |
| SMA | Smooth muscle actin | Blood vessels/endothelial cells | ||
| GFAP | Glial fibrillary acidic protein | Glia | GFAP | |
| NeuN | Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3 | Differentiated nerve | NeuN | |
| LEU7/HNK1/B3GAT1/CD57 | Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 | Schwann cells | B3GAT1 | |
| XBRA | Xbrachyury | Mesoderm during gastrula stage | Brachyury | |
| MSX1 | Muscle segment homeobox, Msh homeobox 1 | Undifferentiated cells | Transcriptional repressor during embryogenesis | MSX1 |
| Trypan blue | Dead tissues or cells | Vital stain | Trypan blue | |
| MEF2C | Myocyte-specific enhancer factor 2C | Differentiated muscle cells | aka MADS box transcription enhancer factor 2, polypeptide C | MEF2C |
| PH3 | Phospho-Histone H3 | Mitotic cells | ||
| BrdU | Bromodeoxyuridine | Cells in S-phase | BrdU | |
| CASP3 | Caspase 3 | Dying cells | CASP3 | |
| GDF5 | Growth/differentiation factor 5 | Joints | Joint development marker | GDF5 |
| Survivin | Cleavage furrow | aka baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5 | Survivin | |
| MAP2 | Microtubule-associated protein 2 | Neurons | MAP2 | |
| SOX9 | SRY (sex determining region Y)-box 2 | Cartillage progenitors | SOX9 | |
| MBP | Myelin basic protein | Myelin sheath | MBP |
Digital data
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.