Please find the following media preparation method:
BCIP/NBT Alkaline Phosphatase Substrate Solution Blue
0.02% BCIP and 0.03% NBT in 0.1M TBS, pH 9.5
1% BCIP (5-bromo-4-chloro-3-indolyl phosphate) in 100%
DMF (dimethylformamide) (light sensitive so store in
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.
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
5 ml AP buffer (pH 9.5)
Incubate sections for 30 min ~ 24 hours at room