X-gal staining of embryos
Protocol supplied by S. P. Yee (firstname.lastname@example.org) and Joe Chan (email@example.com).
- 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.
- 10% NP40
- 1% Deoxycholate (0.5M EGTA (make 1M and dilute with 4M NaOH till dissolves. Check pH=neutral, make up volume for 0.5M)
- 0.5M K3Fe (CN)6 = 1.65 g/10 ml (red)
- 0.5M K4Fe(CN)6 = 2.10 g/10ml (yellow)
- 40 mg/ml X-gal (in dimethylformamide)
- 1M MgC12
- 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 Ál
- 2 mM MgC12 80 Ál
- 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