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Chromogenic Substrates

Fast red (FR) is the most common chromogen used in anatomic pathology systems and gives a very bright magenta or fuchsin-red color. FR has a variety of names associated with it, such as TR/naphthol AS-MX. Some FR compounds are more stable and resist fading in organic solvents, while other FR preparations of are very sensitive to alcohol and xylene and the generated signal will be lost. Technologists overcome this issue by completely air drying the tissue slides and then applying a cover slip with a permanent mounting media. Aqueous based mounting media is a viable option to overcome fading.
FR chromogenic substrate typically comes in tablet form or liquid concentrate. Either form is added to the appropriate volume of napthol phosphate substrate buffer and applied to the tissue sections for the desired reaction time. This time can vary between manufactures, but is usually in the 5, 10, to 20 minute range. It is good laboratory practice to keep the FR chromogenic substrate refrigerated until mixed with room temperature buffer. Once mixed, working FR has a very short bench life and should be used immediately.
Alternate colors can be obtained from chemicals other than FR, such as an indigo blue color that can be obtained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). Other colors such as magenta, blue, brown-black, green, and orange can be obtained from vendors who offer specialty chromogens for IHC.
One word of caution, ALWAYS use Tris buffered saline (TBS) when performing anatomic pathology IHC. Phosphate buffered saline (PBS) will cause the reaction to fail.