Alkaline Phosphatase Substrate-Chromogen (BCIP/NBT) Kit

BCIP/NBT Chromogen

Conc. Buffer 6 mls
Conc. Chromogen 3 mls
Conc. Substrate 3 mls

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)/nitroblue tetrazolium (NBT) substrate for alkaline phosphatase-based detection systems, is a commonly used substrate chromogen. BCIP acts as the substrate for alkaline phosphatase, and the NBT enhances the purplish-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.*

BCIP/NBT is compatible with organic solvents so that it can be counterstained with Nuclear Fast Red or Methylene Green. The slides can be dehydrated and mounted with Clariøn (Cat. No. M05, M06, M07) or other organic solvent-based mounting medium.
For better contrast counterstaining, the BC-50 Red counterstain (Cat. No. M11) can be used. Slides should be mounted in Situ/Mount (Cat. No. M04) when BC-50 Red is used as this counterstain is not compatible with other mounting media.

Refrigerate at 4°C. Do not freeze.

Working Chromogen Preparation: Remove the BCIP/NBT Chromogen kit from the refrigerator 20 minutes before the preparation of the working chromogen solution.
1Transfer 5 mL of distilled water into supplied plastic bottle.
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/NBT 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 addition of the Concentrated Chromogen. Discard unused reagent according to local and state regulations.Avoid contact with skin and clothes.