BCIP/NBT

Application: IHC, ICC and in situ hybridization 1. 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 37ºC 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.

Our unique BCIP/NBT is an excellent one component chromogen substrate system that is used in Immunohistochemistry (IHC), Immunocytochemistry (ICC), western blot (WB) and in situ hybridization with alkaline phosphatase (alk phos). Alkaline phosphatase catalyzes the hydrolysis of a variety of phosphate containing substances in the alkaline pH range. This chromogen system gives purple blue color; is compatible with nuclear fast red (AR-6524) counterstain and can be mounted with aqueous mounting medium or our ImmunoHistoMount (AR-6503) This chromogen can also be counterstained with our Red counter stain (AR-6523) and mounted with mounting medium Immuno In Situ Mount (AR-6507)