The IHC procedure involves several key steps: 1. Fixation: Tissue samples are fixed to preserve cellular structure and protein integrity. 2. Embedding: Fixed tissues are embedded in paraffin to facilitate sectioning. 3. Sectioning: Thin tissue sections are cut and mounted on slides. 4. Deparaffinization and Rehydration: Paraffin is removed, and tissues are rehydrated. 5. Antigen Retrieval: Techniques such as heat-induced epitope retrieval (HIER) or enzymatic digestion are used to expose antigens. 6. Blocking: Non-specific binding sites are blocked to prevent background staining. 7. Primary Antibody Incubation: Tissues are incubated with a primary antibody specific to the target antigen. 8. Secondary Antibody Incubation: A secondary antibody, conjugated with a detection system, binds to the primary antibody. 9. Detection: Common detection systems include chromogenic substrates (e.g., DAB) or fluorescent dyes, which produce a visible signal. 10. Counterstaining and Mounting: The tissue is counterstained (e.g., with hematoxylin) to provide contrast and then mounted for examination.
