INT/BCIP Stock Solution

BCIP (5-bromo-4-chloro-3-indoyl phosphate) is the AP (alkaline phosphatase)-substrate which after dephosphorylation reacts further to give a dark-blue indigo dye as an oxidation product. INT (iodonitrotetrazolium) is the oxidant producing a red dye. The reaction product has a reddish-brown color and is insoluble in water.

INT/BCIP Stock Solution is used for the sensitive detection of alkaline phosphatase (AP) in blotting protocols, that includes:

• Southern blot
• Western blot
• immunohistochemistry and immunocytochemistry

It has been used for colorogenic in situ hybridization.

BCIP is the AP-substrate which after dephosphorylation reacts further to give a dark-blue indigo dye as an oxidation product. INT is the oxidant producing a red dye.

Formulas: INT: C₁₉H₁₃CIN₅O₂; BCIP: C₈H₆NO₄BrCIP x C₇H₉N

Solution of 33 mg/ml INT (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride) and 33 mg/ml BCIP (5-bromo-4-chloro-3-indolyl-phosphate, toluidine-salt in DMSO)

Working solution: Preparation of 10 ml Staining Solution
Bring the stock solution to 15 to 25 °C until all components are dissolved.
Add 75 μl of the stock solution to 10 ml 0.1 M Tris-buffer, pH 9.5, 0.05 M MgCl₂, 0.1 M NaCl.
Note: Prepare the staining solution shortly before use.
Preparation of Additional Solutions Required
Blocking solution: Dissolve 0.5 g Blocking Reagent in 100 ml TBS, pH 7.5, by heating to 50 to 60 °C (1 hour). Dissolving the components can be accelerated by sonication or by incubation in a microwave oven.
Information Note: The solution remains turbid.

For life science research only. Not for use in diagnostic procedures.