An immunohistochemical alkaline phosphatase staining method is an enzyme that hydrolyzes the substrate to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes. Several different combinations of substrates and chromogens have been used successfully.
The purpose of the substrate/chromogen is to use a combination that will produce a color which can be visualized via light microscopy. Fast red TR and fast blue BB (red and blue) produce a bright red or blue end product, but require aqueous mounting media. An alternative to fast red TR and fast blue BB is new fuchsin (red) which is insoluble in alcohol and solvents. Additional substrates include naphthol AS-BI phosphate, naphthol AS-TR phosphate and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP).
Take for example naphthol-AS-phosphate and a chromogen, fast red. Slides can be air dried before coverslipping using either an aqueous or synthetic resin coverslipping medium, depending on the type of chromogen used. It's important to remember that alkaline phosphatase is sensitive to heat and light, which can lead to staining inconsistency. The image displays Varicella zoster in a section of skin, demonstrated with an alkaline phosphatase detection system and fast red chromogen.
