Human Alkaline Phosphatase staining of transgenic embryo/tissue
AP Buffer:
100 mM Tris-HCl, pH 9.5
100 mM NaCl
10 mM MgCl2
PTM:
PBS
0.1% Tween-20
2 mM MgCl2
Since this is generally done in conjunction with lacZ staining, embryo/tissue is usually fixed as per lacZ staining. For wholemount staining, penetration of stain may be improved by fixing in PBS + 0.2% glutaraldehyde + 0.02% NP-40 + 0.01% NaDC (at 4oC).
Rinse in PBS.
Heat inactivate endogenous alkaline phosphatase activity by incubation in PBS at 70-75oC for 30 minutes.
Rinse in PBS at RT.
Wash in AP Buffer for 10 minutes.
Stain with BM Purple AP Substrate at 4oC for 0.5 to 36 hours (N.B. Incubation at RT will give a quicker result but may result in seepage of staining. Also: NBT/BCIP AP staining has been tried with 9.5dpc embryos but gave a mauve background).
Wash extensively in PTM.
