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 1 microliter of INT stock and 4 microliters 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. 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.

Notes

• Do not dehydrate INT/BCIP stained embryos in alcohol or other organic solvents. The stain will be washed away in these organic solvents.

Recipes

• AP buffer: 0.1M Tris pH 9.5; 0.05M MgCl₂; 0.1M NaCl; 0.1% Tween-20
• INT stock: 50 mg/ml 2-(4-iodophenyl)-5-(4-nitrophenyl)-3-phenyltetrazolium (Sigma I8377) in dimethyl sulfoxide (DMSO)
• BCIP stock: 12.5 mg/ml 5-bromo-4-chloro-3'-indolyphosphate (Sigma B6149) in H₂O