IHC procedure

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Stains & CD markers
Immunohistochemistry (IHC) procedure

Author: Nat Pernick, M.D.

Last author update: 1 July 2015

Last staff update: 21 June 2021

Copyright: 2003-2024, PathologyOutlines.com, Inc.

PubMed Search: immunohistochemistry procedure

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Cite this page: Pernick N. IHC procedure. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/stainsihcprocedure.html. Accessed December 25th, 2024.

Definition / general

Immunohistochemistry (IHC) is a tool for surgical pathology and research
Diagnosis should be based on H&E morphology, with confirmation by immunohistochemistry or molecular testing; it is dangerous to use immunohistochemistry alone to make the diagnosis
A stain / result is not just positive or negative; focus on the types of cells that are immunoreactive and determine if they are tumor cells, inflammatory cells, normal cells or stromal cells; comparing the results to an H&E stained section or a negative control of the same block may be helpful (Am J Surg Pathol 2007;31:1627, J Clin Pathol 2011;64:466)
After you identify the type of cell staining, it is helpful to note the percentage of these cells staining, the intensity of staining (weak, 1+, 2+, 3+, 4+) and the pattern of staining (membranous, cytoplasmic, nuclear, dot-like)
The pattern of immunoreactivity should follow the anatomic distribution of the antigen before it is called positive / immunoreactive
Reference: CAP Laboratory Improvement Programs: Principles of Analytic Validation of Immunohistochemical Assays
Antibodies are often useful beyond their recommended expiration dates
Combining results from different studies may be hazardous, as studies may use different antibodies and different standards of interpretation
Recommended to interpret immunohistochemical stains in small needle core biopsy specimens based on the area with the greatest immunoreactivity (Am J Clin Pathol 2007;127:273)

Common errors

Not using a positive or negative control; they are helpful in interpreting the staining pattern, particularly if it is heavy or weak
Other sources of error are ectopic antigen expression (may be due to abundant endogenous biotin, Hum Pathol 2011;42:369), cross reactions (Mod Pathol 2012;25:231), less specificity than thought (Int J Clin Exp Pathol 2012;5:137), use of the wrong secondary antibody or rarely the wrong primary antibody

IHC procedure - 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, NordiQC

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