Alkaline Phosphatase Substrate-Chromogen (BCIP/INT) Kit
Alkaline Phosphatase based immunostaining systems are gaining wide acceptance among pathologists because of their relatively high signal to noise ratio. 5-bromo,4-chloro,3-indolylphosphate BCIP)/iodoblue tetrazolium (INT) substrate for alkaline phosphatase-based detection systems, is a commonly used substrate chromogen. BCIP acts as the substrate for alkaline phosphatase, and the INT enhances the yellow-brown final color of the precipitates at the reaction sites. This chromogen is useful for immunohistochemistry, immunocyto-chemistry and in situ hybridization procedures in both manual and automated systems.*Slides should be counterstained with Biømeda Aqueous Hematoxylin (Cat. Np. M10) and mounted
in Crystal/Mount (Cat. No. M02). BCIP/INT is not compatible with organic solvents.
Refrigerate at 4°C. Do not freeze.
Working Chromogen Preparation: Remove the BCIP/INT Chromogen kit from the refrigerator 20 minutes before the preparation of the working chromogen solution.
1. Transfer 5 mL of distilled water into supplied plastic bottle.
2. Add 2 drops of Concentrated Buffer Reagent. Mix.
3. Add one drop of Concentrated Chromogen Reagent. Mix thoroughly.
4. After chromogen dissolves, add one drop of Concentrated Substrate Reagent. Mix well.
5. Apply BCIP/INT working solution to the specimens. Before applying this solution make sure to wash the specimens extensively with buffer to remove tracing reagent containing alkaline phosphatase.
6. Incubate for 15-30 minutes at room temperature. Wash specimens with distilled water and proceed with the counterstaining steps.* For automated immunohistochemistry, use corresponding number of drops of each reagent according to amount of water needed in the individual procedure.Use the working chromogen solution within 4-6 hours from the time of the additoin of the Concentrated Chromogen. Discard unused reagent according to local and stae regulations.