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 µl
• 2 mM MgC12 80 µl
• 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