BCIP/NBT Alkaline Phosphatase Substrate Solution Blue

Tony Rook

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Nov 16, 2015
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Please find the following media preparation method:

BCIP/NBT Alkaline Phosphatase Substrate Solution Blue


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.


Working Solution:

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.


Link Reference:
http://www.ihcworld.com/_protocols/chromogen_substrates/AP_BCIP_NBT_blue.htm