Immunohistochemistry (IHC) procedure

Author: Nat Pernick, M.D. (see Authors page)

Revised: 28 August 2015, last major update July 2015

Copyright: (c) 2003-2015,, Inc.

PubMed Search: Immunohistochemistry+procedure
  • Most important steps in immunohistochemistry are selection of appropriate antibodies, correct interpretation, technical quality and integration of results into final diagnosis (Am J Surg Pathol 2002;26:873)
  • Pretreatment, often with microwaving of tissue in citrate buffer to unmask antigens hidden by formalin cross-links or other fixative
  • Other agents for pretreatment (antigen retrieval) are pepsin, proteases and trypsin
  • Apply primary antibody (monoclonal antibodies usually are more specific); antibody binds to antigens of interest
  • Wash off excess primary antibody
  • Add biotinylated anti-IgG antibody (secondary antibody), which binds to the primary antibody present
  • Add avidin-biotin-peroxidase complex, which binds to secondary antibody
  • Add 3, 3’ diaminobenzidine (DAB) as a chromagen (color changing reagent), with hematoxylin counterstaining
  • Other enzyme complexes besides avidin-biotin are horseradish peroxidase, alkaline phosphatase with naphthol phosphate and glucose oxidase with nitroblue tetrazolium
  • Other chromagens besides DAB are AEC (water soluble, sensitive to light)
  • Alternative fixation methods are possible, but must validate IHC for each target (Virchows Arch 2012;461:259)
  • References: Wikipedia, IHC World-protocols

Direct method (uses only one antibody)

Indirect method (uses primary and secondary antibodiesy)

Uses by Pathologists
  • Assist with diagnosis, by identifying staining patterns characteristic of specific tumors or disease processes
  • Assist with identification of normal tissue
  • Identify protein overexpression (HER2, EGFR), which is associated with response to treatment, and so is useful to clinicians
  • For research, to assist in understanding disease processes
Micro Images

Membranous staining pattern:
stain localizes along cell membrane

Cytoplasmic staining pattern (left-inhibin, right-beta hCG): stain is diffuse within cytoplasm

Dot-like staining pattern

Golgi staining pattern: prolactin

Nuclear staining pattern: ER in invasive ductal carcinoma

Nuclear staining pattern: p63 in myoepithelial carcinoma