Procedures

1. Rinse immunolabeled embryos twice in AP buffer, 1 minute each
2. Incubate embryos in AP buffer for another 5 minutes.
3. Prepare staining solution while incubating the embryos. Add 4 microliter of NBT stock and 1 microliter of BCIP stock to 250 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. Excessive light exposure turns the specimen reddish due to the photochemical reaction. Check at 15 minutes, 30 minutes, 1 hour, 2 hours and 3 hours after staining solution was added.
6. If desired staining intensity is reached, stop color reaction by washing embryos several times with PBS + 0.1% Tween 20.

Notes

• Extended color reaction produce higher background. In principle, one can continue color reaction in 4°C overnight. But, this results in a higher background and magnesium salt precipitation also starts to build up. It is recommended that if NBT/BCIP fails to produce desirable stain in 3 hours, one can simply use BM Purple instead.