1. Fix embryos in fixing solution (0.5% glutaraldehyde,
1.25 mM EGTA, 2 mM MgCl2, in PBS). For large
embryo, include 2-4% paraformaldehyde.
2. Fix for 15-30 minutes on ice for small embryos, 30 mintes to several hours for large embryos.
3. Rinse with PBS for 15-30 minutes with several changes of PBS.
4. Stain with PBS containing
1 mM MgCl2,
5 mM K4[FeII(CN)6],
5 mM K3[FeIII(CN)6],
0.02% NP-40, 0.01% Na deoxycholate, plus
1 mg/ml X-gal. X-gal staining solution should be warmed to 37 °C for 5-10 minutes before use.
6. Incubate at room temperature or 37 °C in dark until desired level of
staining is observed (several hours to overnight). Stop staining by washing several times with PBS.
7. Store in PBS at 4 °C.