The peroxidase, anti-peroxidase (PAP) method of IHC staining is indirect. When initially developed, it was the first practical method of applying antibodies to paraffin-embedded tissue sections. This method has a high degree of sensitivity, specificity, and stability.
The PAP method employs an unlabeled primary antibody "bridged" to a PAP complex by a secondary antibody. The PAP complex consists of antibodies against horseradish peroxidase and horseradish peroxidase antigen. This complex must be from the same species as the primary antibody. However, the "bridging" secondary antibody is derived from a different species specific to the primary antibody.
Below is a general example of the PAP method of staining:
- Primary antibody specific for the antigen of interest is made in rabbits and reacts with the antigen.
- An excess of “bridging” secondary antibody specific for the immunoglobulin (Ig) of the primary antibody, such as goat anti-rabbit IgG, is applied so that one of the antibody-binding sites reacts with the primary antibody, and the other site remains accessible.
- The PAP complex (a soluble complex consisting of two antibody molecules [derived from the same species as the primary antibody...rabbit in this scenario], three molecules of peroxidase, and two molecules of anti-peroxidase) binds irreversibly to the secondary antibody.
- This reaction is then visualized using hydrogen peroxide as an enzyme substrate and a chromogen such as 3,3’-diaminobenzidine tetrahydrochloride (DAB), resulting in the deposition of the visible reaction product.
