When 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)/Nitroblue Tetrazolium (NBT) is added to tissue sections containing Alkaline Phosphatase (AP) labeled probes, the hydrolysis of the BCIP phosphate group by AP results in the formation of powerful reduced substances. These substances reduce NBT to insoluble NBT formazan products, which is readily visible as fine purple deposits at sites of AP activity. ABM’s BCIP/NBT Dye for Immunohistochemistry provides the greatest sensitivity amongst the immunohistochemistry dyes.
1. Complete all required incubations with antibodies, probes, and AP labeled reagents. 2. Wash tissues sample with Tris-HCl or Tris buffered saline containing 0.1% Tween 20. Note: DO NOT USE phosphate buffers. Inorganic phosphate is a powerful inhibitor of Alkaline Phosphatase. 3. After the final wash, completely cover the sample in the dye solution and incubate for 5-15 minutes. Protect the reaction from light. 4. Stop the enzyme reaction by thoroughly washing the sample with dH2O. Presence of excessive background staining indicates incomplete removal of non bound AP. Increase the number of washes or the washing time to remedy the problem. 5. If desired, counterstain the sample using a 0.5% solution of neutral red prepared in a 0.1M acetate buffer, pH 5.2.
Suitable for the immunohistochemical localization of Alkaline Phosphatase (AP) labeled probes. This dye may also be used for In Situ hybridization and blotting techniques.
Store at room temperature (17-28ºC). Storage at refrigerator temperatures (2-4ºC) will not inhibit product performance but warm the product to reaction temperatures prior to use. Discard if solution turns purple or turbid.
Depending on the enzyme activity, it may be necessary for the tissue sample to undergo longer incubation periods in ABM’s BCIP/NBT dye solution. If colour develops immediately after the start of the incubation period, the formazan deposits may flake off of the tissue sample. To avoid this problem, further dilution of the AP probe is necessary. A fine line of formazan deposits circumscribing the band or dot, with no deposit in the center, also suggests a need for further dilution of the AP probe.