BCIP/NBT Membrane Alkaline Phosphatase Substrate is a useful detection reagent for ELISA, IHC, and in situ hybridization. The substrate is ready to use. To use: Wash the membrane after incubation with the alkaline phosphatase conjugate secondary reagent. Apply sufficient substrate to cover the membrane. Color (purple-turquise, varies with pH) will develop within 30 minutes. Wash, dry membrane and image.