Immunohistochemistry and Molecular Techniques
IHC fundamentals
Immunohistochemistry uses antibodies to detect antigens; molecular methods detect nucleic acids. Preanalytical variables dominate results: cold ischemia, fixation type and duration, and processing. Antigen retrieval reverses formalin cross-linking-heat-induced epitope retrieval (HIER) in citrate pH 6 or EDTA pH 9, or enzymatic protease digestion. Under-retrieval causes false negatives; over-retrieval causes background and false positives. Block endogenous peroxidase with hydrogen peroxide and block nonspecific binding with protein. Avidin-biotin detection is vulnerable to endogenous biotin in liver and kidney; polymer detection avoids this.
Controls and interpretation
Include a positive tissue control and a negative reagent control, ideally on the same slide or run. Titrate antibodies and verify each new lot. Common chromogens are DAB (brown) and Fast Red; hematoxylin is the usual counterstain. Molecular tests include ISH (e.g., EBER), FISH (e.g., HER2), PCR, and sequencing; all require adequate cellularity and fixation.
Traps and a worked example
Necrotic edges, crush artifact, and overfixation mimic negative staining. Do not call any brown pigment positive; endogenous pigment and DAB precipitate can mislead.
Worked example: HER2 IHC scored 2+ is equivocal and requires ISH for gene amplification; 3+ is positive and 0/1+ is negative. A weak stain without a valid positive control is not interpretable.