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Stains

Immunohistochemistry (IHC) Procedure

Reviewer: Nat Pernick, M.D., PathologyOutlines.com, Inc. (see Reviewers page)
Revised: 20 April 2011, last major update April 2011
Copyright: (c) 2002-2011, PathologyOutlines.com, Inc.

General
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1. Pretreatment, often with microwaving of tissue in citrate buffer to unmask antigens hidden by formalin cross-links or other fixative
2. Other agents for pretreatment (antigen retrieval) are pepsin, proteases, trypsin
3. Apply primary antibody (monoclonal antibodies usually are more specific); antibody binds to antigens of interest
4. Wash off excess primary antibody
5. Add biotinylated anti-IgG antibody (secondary antibody), which binds to the primary antibody present
6. Add avidin-biotin-peroxidase complex, which binds to secondary antibody
7. 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)
● Most important steps are selection of appropriate antibodies, correct interpretation, technical quality and integration of results into final diagnosis (Am J Surg Pathol 2002;26:873)

Diagrams
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Direct method (uses only one antibody)


Indirect method (uses primary and secondary antibodiesy)

Uses
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● 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 - staining patterns
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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 - estrogen receptor stain is diffuse within nucleus

Additional references
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● Wikipedia, IHC World-protocols

End of Stains > Immunohistochemistry (IHC) Procedure

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