When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique

Vet Pathol. 2014 Jan;51(1):42-87. doi: 10.1177/0300985813505879. Epub 2013 Oct 15.


Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

Keywords: antibodies; antigen retrieval; fixation; immunohistochemistry; review; standardization; technical aspects; validation.

Publication types

  • Review

MeSH terms

  • Animal Diseases / diagnosis*
  • Animals
  • Antibodies
  • Antigen-Antibody Reactions
  • Antigens
  • Immunohistochemistry / methods
  • Immunohistochemistry / standards
  • Immunohistochemistry / veterinary*
  • Pathology, Veterinary / methods*
  • Sensitivity and Specificity
  • Tissue Array Analysis / veterinary
  • Tissue Fixation / veterinary


  • Antibodies
  • Antigens