Immunohistochemistry (IHC)

Principle

• Specific antibodies bind target antigens in tissue sections; the binding is made visible by an enzyme label and chromogen.
• Modern methods use polymer-based detection with horseradish peroxidase (HRP) and DAB (brown product) or alkaline phosphatase with a red chromogen.
• Polymer systems avoid false staining from endogenous biotin seen with older avidin–biotin methods.

Specimen

• FFPE sections (about 3–4 µm) on positively charged slides; cytology cell blocks; well-controlled fixation (e.g. 10% NBF).

Reagents and equipment

• Antigen retrieval buffers (citrate pH 6, or Tris–EDTA pH 9), hydrogen peroxide block, protein block, primary antibodies, detection kit, chromogen, hematoxylin counterstain.
• Automated immunostainers are widely used.

Procedure

• Dewax and rehydrate sections.
• Heat-induced epitope retrieval (HIER) reverses formalin cross-links; some antibodies use enzyme retrieval.
• Block endogenous peroxidase (e.g. 3% H2O2) and non-specific binding.
• Apply primary antibody, then the detection system and chromogen, with buffer washes between steps.
• Counterstain, dehydrate, clear and mount.

Results

• Positive signal is brown (DAB) or red in the expected location: nuclear (e.g. ER, Ki-67), cytoplasmic (e.g. cytokeratin) or membranous (e.g. HER2, CD20).
• Scoring systems apply for predictive markers (e.g. HER2 scored 0 to 3+).

Quality control and pitfalls

• Positive tissue controls (ideally on the same slide) and negative reagent controls; internal controls (e.g. normal breast ducts for ER) confirm the run.
• Poor fixation, strong acid decalcification and drying of sections cause false negatives or artifacts.
• Edge artifact, endogenous peroxidase, pigment (melanin, hemosiderin) and cross-reactivity cause false positives.
• New antibodies and methods must be validated before clinical use.

Clinical use

• Tumor lineage (e.g. cytokeratins for carcinoma, CD45 for lymphoma, S100/SOX10 for melanoma, desmin for muscle).
• Site of origin (e.g. TTF-1, CDX2, CK7/CK20 pattern).
• Predictive and prognostic markers (ER, PR, HER2, PD-L1, Ki-67, mismatch repair proteins) and infectious agents (e.g. CMV, H. pylori).