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].

Guidelines (The following is our recommended experimental protocol. This protocol is for reference only, and specific operations should be adjusted according to your actual needs).
NBT and BCIP staining protocol
1. Materials
Dimethylformamide (DMF, HY-Y0345).
Nitro blue tetrazolium chloride (NBT, HY-15925) stock solution: Dissolve 0.5 g NBT in 10 mL 70% aqueous DMF.
BCIP stock solution: Dissolve 0.5 g BCIP in 10 mL 100% DMF.
Substrate Buffer: 100 mM diethanolamine buffer (pH 9.5) containing 100 mM NaCl and 5 mM MgCl₂.
2. Methods
2.1 To prepare the NBT/BCIP solution, add 66 μL NBT stock solution to 10 mL Substrate Buffer and mix thoroughly. Add 33 μL BCIP stock solution to the mixture. Use the NBT/BCIP solution within 1 hour.
2.2 Place the blot into an appropriate container and add the NBT/BCIP solution. Use 10 mL solution for a 15 × 15 cm² membrane.
Agitate at room temperature and incubate the blot until the bands develop to the desired darkness. Typically, about 30 minutes is required.
2.3 Remove the NBT/BCIP solution and rinse the blot with water to stop the reaction.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

• [1]. Trinh le A, et al. Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain. Biotechniques. 2007 Jun;42(6):756-9.  [Content Brief]