Nitro blue tetrazolium chloride

Nitro blue tetrazolium chloride (NBT) is a substrate for dehydrogenases; is used with the alkaline phosphatase substrate 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) in western blotting and immunohistological staining procedures^[1]. In Vitro:Protocol for Staining with NBT and BCIP
1.Materials
Dimethylformamide (DMF, HY-Y0345).
Nitro blue tetrazolium chloride (NBT, HY-15925) Stock Solution: Dissolve 0.5 g of NBT in 10 mL of 70% DMF in water.
BCIP Stock Solution: Dissolve 0.5 g of BCIP in 10 mL 100% DMF.
Substrate Buffer: 100 mM diethanolamine buffer (pH 9.5) containing 100 mM NaCl and 5 mM MgCl₂.
2.Method
2.1 To prepare the NBT/BCIP solution, add 66 µL of stock NBT to 10 mL of substrate Buffer and mix well. Add 33 µL of stock BCIP to the solution. Use the NBT/BCIP solution within 1 hour.
2.2Place blot in a suitable container and add the NBT/BCIP Solution. Use 10 mL of solution for a 15 × 15cm² membrane.
Incubate blot at room temperature with agitation until the bands are suitably dark. Typically, ~30 minutes is required for development.
2.3 Remove the NBT/BCIP Solution and rinse blot with water to stop the reaction.