Immunohistochemistry and Enzyme Histochemistry
Preanalytical variables
IHC depends on antibody binding an epitope, visualized by a chromogen. The dominant variables are preanalytical: cold ischemia time, fixative type and fixation duration. NBF for 6-48 hours is the validated window for most predictive markers (ER, PR, HER2, Ki-67); overfixation masks epitopes, underfixation causes cytoplasmic blush and false positivity.
Retrieval and detection
Antigen retrieval is heat-induced epitope retrieval (HIER) in citrate pH 6.0 or Tris/EDTA pH 9.0, or enzymatic digestion with trypsin or proteinase K. The retrieval condition must match the antibody clone.
Detection is polymer based (compact and sensitive) or avidin-biotin complex. Peroxidase systems need a peroxide block to quench endogenous peroxidase in red cells and neutrophils; ABC systems need a biotin block in kidney and liver. Chromogens: DAB is brown and permanent; AEC and Fast Red are red and alcohol soluble, requiring aqueous mounting.
Controls
Include a positive tissue control, a negative reagent control of the same isotype, and for mouse tissue a mouse-on-mouse block, since endogenous mouse immunoglobulin binds the secondary antibody.
Enzyme histochemistry
Enzyme histochemistry detects enzyme activity rather than antigen and generally requires unfixed or lightly fixed frozen sections because processing destroys enzymes: examples include myeloperoxidase, nonspecific esterase and NADH.
Traps: reading edge artifact or necrosis as true positivity; ignoring endogenous biotin or peroxidase; running IHC on acid-decalcified bone; grading a marker without a validated control.
Rule of thumb: If the control is negative the run fails; repeat before interpreting the patient slide, however convincing the tumor looks.