Combination of NBT (nitro blue tetrazolium chloride) and BCIP (5-brom-4-chloro-3'-indolyphosphate p-toluidine salt) makes it possible to obtain an intense black violet insoluble precipitate when it reacts with alkaline phosphatase, an enzyme widely used for immunoassays such as IHC, WB or ELISA.

These substrates thus permit detect alkaline phosphatase activity on a solid surface, such as nitrocellulose membranes or tissue sections.

The precipitate produced is partially insoluble in alcohol. This combination of substrates is more sensitive than other substrates of alkaline phosphatase such as AP-Red or AP-Blue.