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 4o C).

• Rinse in PBS.
• Heat inactivate endogenous alkaline phosphatase activity by incubation in PBS at 70-75o C for 30 minutes.
• Rinse in PBS at RT.
• Wash in AP Buffer for 10 minutes.
• Stain with BM Purple AP Substrate at 4o C 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.