1. Fix embryos in fixing solution ( 0.5% glutaraldehyde, 1.25 mM EGTA, 2 mM MgCl₂, 0.02% NP-40, 0.01% Na deoxycholate, in PBS). For large embryo, include 2-4% paraformaldehyde.

2. Fix for 15-30 minutes on ice for small embryos, 30 minuets to several hours for large embryos.

3. Rinse with PBS for 15-30 minutes with several changes of PBS.

4. Heat inactivate endogenous alkaline phosphatase (AP) by incubation in PBS at 70-75 °C for 30 minutes.

5. Rinse with PBS.

6. Rinse in 100 mM Tris-HCl (pH9.5), 100 mM NaCl, 10 mM MgCl₂ for 10 minutes.

7. Stain in 100 mM Tris-HCl (pH9.5), 100 mM NaCl, 10 mM MgCl₂, 0.02% NP-40, 0.01% Na deoxycholate, plus 0.337 mg/ml NBT and 0.174 mg/ml BCIP.

8. Incubate at room temperature or 4 °C in dark until desired level of staining is observed (10 minutes to overnight).

9. Stop staining by washing several times with PBS containing 0.1% Tween-20 and 2 mM MgCl₂.

10. Store in PBS at 4 °C.