Immunohistochemistry (IHC) procedure

Topic Completed: 1 July 2015

Revised: 25 February 2020

Copyright: 2003-2020,, Inc.

PubMed Search: immunohistochemistry procedure

Nat Pernick, M.D.
Page views in 2019: 2,542
Page views in 2020 to date: 581
Cite this page: Pernick N. Immunohistochemistry (IHC) procedure. website. Accessed February 27th, 2020.
Definition / general
  • 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
Diagrams / tables

Images hosted on other servers:

Direct method (uses only one antibody)

Indirect method (uses primary and secondary antibodies)

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
Microscopic (histologic) images

Case of the Week #200

Cytoplasmic staining pattern (beta hCG):
stain is diffuse within cytoplasm

Images hosted on other servers:

Golgi staining pattern: prolactin

Cytoplasmic staining pattern (inhibin):
stain is diffuse within cytoplasm

Nuclear staining pattern: p63 in myoepithelial carcinoma

Dot-like staining pattern


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