IHC is a multiple-step, diagnostic process that requires the selection and use of appropriate reagents, fixation, processing, slide preparation, and controls. IHC is accomplished via an application of antibodies that recognize a target. There are many methods in current use, but most applications require an enzyme (protein catalyst), a substrate (substance which 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 reaction 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
- Dehydration and mounting with the appropriate media
IHC is considered ancillary testing within anatomic pathology for the identification of tumor antigens, since it is used in conjunction with the morphology information gathered through the evaluation of the H&E stained sections by a pathologist. IHC provides support for an initial diagnosis through both positive and negative findings (binary scoring) for most antibodies.