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X-gal staining of embryos

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1054

 

X-gal staining of embryos

Protocol supplied by S. P. Yee (spyee@julian.uwo.ca) and Joe Chan (cchan@hgmp.mrc.ac.uk).


 

Procedure

  • Dissect embryos in PBS
  • Immediately transfer the embryos to fixative at 4oC, incubate in dark for 1 hour.
  • Rinse the embryos with rinse buffer at room temperature 3 x 15 minutes.
  • Incubate in dark with staining buffer at 37oC 3-7 hours or overnight.

 

Materials

  1. 10% NP40
  2. 1% Deoxycholate (0.5M EGTA (make 1M and dilute with 4M NaOH till dissolves. Check pH=neutral, make up volume for 0.5M)
  3. 0.5M K3Fe (CN)6 = 1.65 g/10 ml (red)
  4. 0.5M K4Fe(CN)6 = 2.10 g/10ml (yellow)
  5. 40 mg/ml X-gal (in dimethylformamide)
  6. 1M MgC12

1.Fixative

  • 4% Paraformaldehyde in PBS

2. Rinse buffer Final concentration Stock

  • 5 mM EGTA 2.0 ml
  • 0.01% Deoxycholate 2.0 ml
  • 0.02% NP40 0.4 ml
  • 2 mM MgCl2 0.4 ml
  • Make up to 200ml with PBS

3. Staining buffer Final concentration Stock

  • 5 mM K3Fe(CN)6 0.4 ml
  • 5 mM K4Fe(CN)6 0.4 ml
  • 5 mM EGTA 0.4 ml
  • 0.01% Deoxycholate 0.4 ml
  • 0.02% NP40 80 �
  • 2 mM MgC12 80 �
  • make up to 39 ml with PBS
  • add 1 ml of stock X-gal solution to make up the final concentration of 1 mg/ml

 

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