Standard IHC staining methods include incubation of slides with a substrate-chromogen complex following labeling steps to visualize the antibody-antigen reaction. This results in a stable, colored end product suitable for visualization with light microscopy. This staining results from the reaction of a soluble substrate with an enzyme to produce an insoluble, colored product. The intensity of staining directly correlates with the concentration of the primary antibody and respective tissue antigen.
Commonly used labeling enzymes for IHC procedures include:
- Horseradish peroxidase (HRP)
- Alkaline phosphatase (AP)
The choice of chromogen used is specific to the labeling enzyme employed for the staining procedure. This is discussed further in the following sections.
