BCIP (BCIP p-toluidine salt) (BCIP p-toluidine salt) is an artificial chromogenic substrate. It is used for the sensitive colorimetric detection of alkaline phosphatase activity.

Instructions
1. Solution preparation:
Stock solution: Prepare BCIP stock solution, usually at a concentration of 50 mg/mL. Dissolve BCIP in an appropriate amount of dimethylformamide (DMF) or water and store at -20°C in the dark for several weeks.
Working solution: Dilute BCIP to the desired concentration (usually 0.15–0.5 mg/mL) in an appropriate substrate buffer. The buffer is usually Tris-HCl (pH 9.5), and MgCl₂ (1–5 mM) is added to enhance enzyme activity.
2. Blotting: Block nonspecific binding after protein, membrane transfer after electrophoresis, or nucleic acid transfer. Add alkaline phosphatase-labeled antibodies or probes, incubate according to the experimental protocol, and wash to remove unbound portions.
Add BCIP/NBT (Nitro Blue Tetrazolium) working solution and incubate in the dark until color development is complete (usually 5–30 minutes). Terminate the reaction with distilled water and dry the membrane for result analysis.
3. Histochemical staining:
After reacting tissue sections or cell samples with ALP-related antibodies or probes, wash them clean.
Add BCIP working solution, incubate until color development is complete, and terminate the reaction with PBS or water.
4. Observe the staining results under a microscope.