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RESEARCH DIVISION Laboratory Manual

 


 

b-Gal Staining of Murine Embryos

Solutions

  1. 500mM K4Fe(CN)6.3H2O (Potassium Ferrocyanide) (Sigma: P9387) in DDW
  2. 500mM K3Fe(CN)6 (Potassium Ferricyanide) in DDW
  3. Solution A : 0.2M NaH2PO4.H2O (27.6g/l)
  4. Solution B : 0.2M Na2HPO4 (28,4g/l)
  5. Phosphate buffer : 23ml of 0.2M NaH2PO4.H2O, 77ml of 0.2M Na2HPO4 and add 100ml DDW. the pH will be approx 7.3
  6. 0.5M EGTA in 0.1M Phosphate buffer, pH increase to 7.
    FW 380.4, so 19.02g for making 100ml of 0.5M EGTA.
    Ethylene Glycol-bis (b-aminoethyl ether)N,N,N',N'-tetraacetic acid) (Sigma E-4378)
  7. Wash buffer : 200ml of wash buffer combine by
    23ml of 0.2M NaH2PO4.H2O,
    77ml of 0.2M Na2HPO4,
    400ml of 1M MgCl2
    2ml of 0.5M EGTA
    0.2ml of 20% NP-40
    0.2ml of 10% Na Deoxycholate
    DDW up to 200ml
  8. Fixing buffer : 198ml 0.1M phosphate buffer,
    400ml of 1M MgCl2
    2ml of 0.5M EGTA
  9. X-gal : 40 mg/ml in diemthylformamide, covered by foil and store at -20C.
  10. 10% formalin (20L)
    95.36g KH2PO4
    192g K2HPO4
    200 ml 40% formadhyde
    the pH should be 7.4

Fixtive

using fix buffer to make 0.2% Glutaraldehyde fixative, eg: add 0.2ml 25% Glutaraldehyde (EM grade) into 24.8ml fixing buffer. (fresh make every time)

For E10 - E12 embryo

- fix the embryo on ice for 10 minutes (with at least 20ml fixative);
- rinse with PBS 2 times;
- wash with 20 ml PBS for 20 min by 3 times;
- pre-stain at 340C for 40 min in 15 ml staining buffer without X-gal;
- add X-gal (final concentration : 500mg/ml) staining at 340C for overnight;
- de-stain and post-fix in 10% formalin
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998