BCIP/NBT

IHC, ICC and in situ hybridization1. After the alkaline phosphatase reaction, wash slide with high pH buffer (~Tris/HCl 9.5).2. Add few drops of ready to use BCIP/NBT reagent, incubate at RT or 37 degree C for 5-15 minutes.3. Wash 2-5X with buffer, followed by rinsing with distilled/deionized water.4. Add counterstain compatible with this chromogen (see description above)5. Wash with tap water, followed by rinsing with distilled/deionized water.6. Mount with aqueous mounting medium (see description above).Western Blot:After alk phos. reaction, wash blots with high pH buffer (Tris ~9.5) containing 0.1 % Tween 20. Incubate membrane for 5-10 minutes or till proper purple blue color bands are observed, protect membrane from light. Wash membrane thoroughly with buffer containing detergent, followed by rinsing with distilled or deionized water.The optimum time of incubation for BCIP/NBT should be determined by the individual lab.Phosphate buffer is a strong inhibitor of alkaline phosphatase and should not be used.