Stains & CD markers
Immunohistochemistry (IHC) procedure

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Last staff update: 21 June 2021

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PubMed Search: immunohistochemistry procedure

Nat Pernick, M.D.
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Cite this page: Pernick N. IHC procedure. website. Accessed October 3rd, 2023.
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

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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