Final Dilution:

        0.02% BCIP and 0.03% NBT in 0.1M TBS, pH 9.5

Stock Solutions:

         1% BCIP (5-bromo-4-chloro-3-indolyl phosphate) in 100% DMF (dimethylformamide) (light   sensitive so store in brown bottle)

       1.5% NBT (nitro blue tetrazolium) in 70% DMF (dimethylformamide) (light sensitive so store in brown bottle. NBT dust is extremely toxic!)

       50mM Levamisole in Distilled Water.

         AP Buffer (100mM Tris-HCl, 100mM NaCl, 5mM MgCl₂, 0.05% Tween 20, pH 9.5):

         Tris-HCl (MW 157.60) ---------------------- 15.8 g

         NaCl (MW 58.44) --------------------------- 5.8 g

         MgCl₂ (MW 203.31) ------------------------ 1.02 g

         Distilled water ----------------------------- 900 ml

         Mix to dissolve. Adjust pH to 9.5 and then bring total volume to 1 liter. Add 0.5 ml Tween 20. Store this solution at room temperature.

       3 drops of 1% BCIP (1 drop = 40 ul).

       3 drops of 1.5% NBT

       2 drops of 50mM Levamisole (To block endogenous AP activity if necessary for frozen sections. Not for paraffin sections since boiling temperature only destroy AP not HRP)

       5 ml AP buffer (pH 9.5)

       Incubate sections for 30 min ~ 24 hours at room temperature.