日本Enzyme-Mediated Detection
When choosing a substrate for conversion by an enzyme, one should select a substrate which yields a precipitating product. Examples of commonly-used substrates are listed below.
| Substrate | Abbrev- iation | Final Color | Soluble in Alcohol (for counter- stain) | Comments |
| Diaminobenzidine | DAB | Brown | No | 3,3'-diaminobenzidine produces a brown end product which is highly insoluble in alcohol and other organic solvents. Oxidation of DAB also causes polymerization. DAB has the ability to react with osmium tetroxide, and thus is very useful in electromicroscopy as well as traditional immunohistochemistry sections. |
| Diaminobenzidine with nickel enhancement | DAB/Nickel | Gray /Black | No | Produces a more intense stain which is resistant to alcohol and provides better contrast, up to 40 times more sensitive than DAB without enhancement |
| 3-Amino-9-ethylcarbazole | AEC | Red/ Brown | Yes | AEC produces a red/brown reaction product Brown and is widely used for immunohistochemical staining. Slide specimens processed with AEC must not be immersed in alcohol or alcoholic solutions (e.g., Harris' hematoxylin). Instead, an aqueous counterstain and mounting medium should be used. AEC is also susceptible to further oxidation when exposed to light and thus it will fade overtime. Dark storage and brief light viewing are recommended. |
| 4-Chloro-1-naphthol | 4-CN | Blue/ Gray | Yes | 4-chloro-1-naphthol (CN) precipitates as a blue Gray end product. Because CN is soluble in alcohol and other organic solvents, the slides must not be dehydrated, exposed to alcoholic counterstains, or coverslipped with mounting media containing organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation, thus it is not usually recommended for Immunohistochemistry but can be used for Western blotting |
| Naphthol AS B1 phosphate/fast Red TR | NABP/FR | Red | Yes | Napthol AS-acts as the substrate for alkaline phosphate/fast red TR phosphatase, and the Fast Red chromogen precipitates at the enzymatic sites producing a vibrant red/pink color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting medium should be used |
| Naphthol AS MX phosphate/fast Red TR | NAMP/FR | Red | Yes | Napthol AS- acts as the substrate for alkaline phosphatase, and the fast red chromogen precipitates at the enzymatic sites producing a vibrant red/pink color. Precipitate is soluable in alcohol thus aqueous counterstain and mounting medium should be used |
| Naphthol AS B1 phosphate/new fuschin | NABP/NF | Red/ Violet | Yes | Napthol AS- acts as the substrate for alkaline phosphatase, and the new Fuchsin chromogen precipitates at the enzymatic sites producing a vibrant red/violet color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting medium should be used. |
| Bromochloroindolyphos- phate/Nitro Blue Tetrazolium | BCIP/NBT | Purple | No | 5-bromo,4-chloro,3-indolylphosphate (BCIP)/ nitroblue tetrazolium (NBT) substrate is a commonly used substrate chromogen. BCIP acts as the substrate for alkaline phosphatase, and the NBT enhances the purplish-brown color of the precipitate. BCIP/NBT is compatible with organic solvents so it can be used with alcohol based counterstains including Nuclear Fast Red or Methylene-Green. |
