Immunohistochemistry and Enzyme Histochemistry
Antigenicity is decided upstream
Ischemic time, fixative, and fixation duration determine whether an antibody can bind. Overfixation creates methylene cross-links that mask epitopes, which is what antigen retrieval attempts to reverse. HIER (heat-induced epitope retrieval) uses buffer - commonly citrate pH 6 or Tris/EDTA pH 9 - in a water bath, steamer, or pressure cooker. Proteolytic retrieval (trypsin, pepsin, proteinase K) digests masking protein and is harsher on morphology. Buffer choice matters: some epitopes require high pH, others low.
Detection and blocking
A primary antibody binds antigen; a secondary or polymer carries the enzyme; a chromogen precipitates. DAB gives a brown, alcohol-insoluble product; AEC gives red but is alcohol-soluble, so it must be coverslipped with an aqueous mountant. Peroxidase systems need a peroxidase block (hydrogen peroxide) to destroy endogenous enzyme, especially in blood-rich tissue, and avidin–biotin systems need a biotin block in liver and kidney. A protein block reduces nonspecific binding. Rinse between steps and never let sections dry - drying creates hard-edged background.
Controls and enzyme histochemistry
Controls are the highest-yield topic: a positive tissue control proves the reagents worked; a negative reagent control (primary omitted or replaced with an isotype-matched irrelevant antibody) proves the signal is specific. Enzyme histochemistry requires enzymes to survive, so fresh, unfixed, snap-frozen tissue is used - ATPase fiber typing, esterase, NADH-TR, acetylcholinesterase for Hirschsprung disease. Fixation and paraffin processing destroy most enzyme activity.
Rule of thumb: brown alone is not positive - accept a signal only when the positive control stains and the negative control does not.