1. After antibody washes, rinse tissues twice for 5 min in alkaline phosphatase buffer (APB): 100 mM Tris, pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, and 5 mM Levamisol. APB should be made fresh just before use, since the levamisol is not stable. Levamisol can be made as a 1 M stock and stored at -20°C.
2. For color development, replace the last wash with APB containing 4.5 ||L NBT (nitro blue tetrazolium, 75 mg/mL in 70% dimethyl formamide) and 3.5 |L BCIP (5-bromo-4-chloro-3-indolyl-phosphate, 50 mg/mL in 100% dimethyl formamide)/ mL. Alternatively, the color reaction can be carried out by placing the tissues directly into BM purple AP substrate, precipitating (BMB cat. no. 1442 074). The BM purple substrate tends to give less background staining than the NBT/ BCIP system, and is recommended if long reaction times are required. The appearance of staining is dependent on the level of gene expression and the probe that is used. Positive staining can become visible after several minutes, or can take up to 1 d.
3. When satisfied with the color development, stop the chromogenic reaction by placing the tissues in MEMFA. This postfixation stops the color reaction and stabilizes the stain. Postfix the tissues for at least 1 h, or they can be left overnight.
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