1. Fix for 15-30 min in 4% paraformaldehyde (4 oC)
2. Wash 3 times in PBS.
If you want to stain for beta gal the do this now, before step 3.
3. Heat the embryos for 40 minn at 45 oC, to inactivate endogenous alkaline phosphatases. Heat in PBS NOT AP staining buffer.
4. Incubate the embryos in Levimisole for 1-2 hours.
- (50x levimisole + 12mg/ml in H2O or staining buffer).
5. Stain the embryos for 1-4 hours.
- AP staining buffer: 100mM Tris-Cl pH 8.5 (or higher?). 100mM NaCl. 50mM MgCl2.
Stocks to add:
- 50x NBT =50mg/ml in 70% dimethylformamide.
- 100x BCIP is 10 mg/ml in dimethylformimide.