5-Bromo-4-chloro-3-indolyl phosphate (BCIP) is a chemical compound used in immunoblotting, in situ hybridization

In situ hybridization

In situ hybridization is a type of hybridization that uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence in a portion or section of tissue , or, if the tissue is small enough , in the entire tissue...

, and immunohistochemistry

Immunohistochemistry

Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...

, with nitro blue tetrazolium chloride

Nitro blue tetrazolium chloride

Nitro blue tetrazolium is a chemical compound composed of two tetrazole moieties. It is used in immunology for sensitive detection of alkaline phosphatase . NBT serves as the oxidant and BCIP is the AP-substrate ....

 (NBT), for sensitive colorimetric detection of alkaline phosphatase

Alkaline phosphatase

Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...

. BCIP is the alkaline phosphatase substrate

Substrate (biochemistry)

In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...

, which is dephosphorylated by the enzyme and then dimerizes. This dimer reduces nitroblue tetrazolim (NBT) to form an insoluble dark blue diformazan precipitate. Alkaline phosphatase is commonly conjugated to secondary antibodies.