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Table of Contents
Definition / general | Diagrams / tables | Uses by pathologists | Microscopic (histologic) images | VideosCite this page: Immunohistochemistry (IHC) procedure. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/stainsihcprocedure.html. Accessed July 12th, 2017.
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
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
Microscopic (histologic) images
Images hosted on Pathout server:
Dot-like staining pattern
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: ER in invasive ductal carcinoma
Nuclear staining pattern: p63 in myoepithelial carcinoma
Videos
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