Procedures

1. Rinse embryos twice in AP buffer, 1 minute each
2. Incubate embryos in AP buffer for 5 minutes to further equilibrate the buffer.
3. Prepare staining solution while incubating the embryos. Add 4 microliter of BCIP stock to 200 microliter AP buffer (scale up if necessary). Mix well by vortexing.
4. Remove AP buffer from embryos, and add staining solution to the embryos.
5. Allow color development in dark. Carry out the color reaction at 4°C if necessary. Check at 15 minutes, 30 minutes, 1 hour, 2 hours and 3 hours after staining solution was added. Continue color reaction in 4°C if necessary.
6. If desired staining intensity is reached, stop color reaction by washing embryos several times with PBS + 0.1% Tween 20.

Recipes

• AP buffer: 0.1M Tris pH 9.5; 0.05M MgCl2; 0.1M NaCl; 0.1% Tween-20
• BCIP stock: 12.5 mg/ml 5-bromo-4-chloro-3'-indolyphosphate (Sigma B6149) in H2O