Immunohistochemistry (IHC) is a multiple-step diagnostic process that requires selecting and using appropriate reagents, fixation, processing, slide preparation, and controls. IHC is accomplished via the application of antibodies that recognize a target antigen. There are many methods in current use, but most applications require an enzyme (protein catalyst), a substrate (a substance that acts on the enzyme), and a chromogen (a substance that is capable of producing a colored end product) at the site of the reaction. This sequence allows visualization of the response at the site of the chromogen deposition under light microscopy.
Protocol steps vary by specific procedure. However, a high-level outline of the basic IHC staining process would include:
- Tissue and slide preparation, including formalin fixation, tissue processing, paraffin embedding, and microtomy
- Deparaffinization and rehydration of tissue sections
- Antigen/epitope retrieval
- IHC staining, including detection and application of the primary antibody
- Counterstaining
- Dehydration and mounting with the appropriate media
IHC is considered ancillary testing within anatomic pathology for identifying tumor antigens since it is used with the morphology information gathered through a pathologist's evaluation of the H&E-stained sections. IHC supports an initial diagnosis through positive and negative findings (binary scoring) for most antibodies.
