ISSN 2398-2950      

Immunohistochemistry (IHC)


Synonym(s): Immunocytochemistry; immunofluorescence; immunoperoxidase


  • Immunohistochemistry (IHC) is a molecular technique which is widely used within veterinary diagnostic laboratories.
  • The technique involves the detection of a particular antigen within a section of tissue via the use of antibodies which bind specifically to that antigen.
  • A similar process can also be used on a cytological sample (eg smear or cytospin) when it is referred to as immunocytochemistry.
  • The antigen-antibody interaction can be visualized via different means such as a fluorescent dye or an enzymatic color change reaction.
  • If the antibody is conjugated to an enzyme such as peroxidase, this enzyme can then catalyze a color-producing reaction (known as immunoperoxidise staining).
  • Alternatively, the antibody can be labelled with a fluorophore and the interaction visualized via a fluorescence microscope (immunofluorescence).


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  • Depending on the sample and the antibody used, some cases will require antigen retrieval pre-treatments prior to the test itself. This is because the fixation and processing of tissues results in cross-linking of some of the proteins in the sample. If extensive, this cross-linking can make the antigen of interest unrecognizable by the antibody.
  • There are various types of antigen retrieval methods including those utilizing heat from a water bath, pressure cooker, steamer or microwave (known as heat-induced epitope retrieval or HIER), enzymatic treatments such as trypsin, or various combinations.
  • IHC involves several steps, the number of which depends on the precise technique used.
  • Regardless, the first step involves blocking of non-specific background staining, often using substances such as skimmed milk or normal serum.
  • Background staining can occur for a number of reasons, including inadequate or delayed fixation of samples (eg in the center of large tissue blocks), or endogenous enzyme activity or biotin within certain tissues.
  • Sections are thoroughly washed between each step in the process; to remove any unbound antibodies or other chemicals.

Direct method

  • A labelled antibody is used which directly detects the antigen within the tissues without the need for any further steps.
  • This technique is the shortest and simplest to perform.
  • However it is relatively insensitive compared to the indirect method.

Indirect method

  • An unlabelled primary antibody is used to detect the antigen within the tissues.
  • This primary antibody is then itself detected by labelled secondary antibody (directed against the species and isotype of the primary antibody).
  • This results in amplification of the signal and thus increased sensitivity of the test.
  • Some other methods involve a third layer; eg in the ABC method the secondary antibody is conjugated to biotin, which then in turn forms a complex with avidin and peroxidase.
  • For those methods using immunoperoxidase, the final step involves incubation of the sample with the chromogenic substrate before visualization by routine light microscopy.
  • The peroxidase catalyzes a color-change reaction; most commonly the substrate-chromagen used is peroxide with diaminobenzidine (DAB), which produces a brown deposit in the tissue sections.
  • By using different colored labels (either color-change or fluorochromes) more than one antigen can be detected in the same tissue section at the same time, which may allow for co-localization of different antigens.


  • Positive and negative controls are always performed.
  • Positive controls test the protocol is carried out correctly (positive control).
  • These may include internal positive control, ie tissue components within the sample itself which would be expected to stain positive with the particular antibody used. For example immunostaining of a skin biopsy for epithelial cell markers would use the epidermis as the internal positive control.
  • External positive controls use another tissue sample which would be expected to stain positive with the particular antibody used, ie a known previously positive-staining tissue.
  • Negative controls test the specificity of any antibody used. Negative controls can either consist of omitting the primary antibody or replacing the primary antibody with an antibody that should not bind to any components of the tissue, in which case no positive staining should be present. This aids differentiation between non-specific and specific staining of cells.



  • The sensitivity of this type of assay is typically high due to amplification of the signal, since multiple antibodies can potentially bind to each antigen.
  • This sensitivity can often be increased depending on the precise technique used.
  • In situ visualization: staining is visualized in situ which is important as it allows correlation of the antigen, be it a cell surface marker or an infectious agent, with a specific cell population and/or lesion.
  • Utilization of biopsy tissues: as the technique uses FFPE tissues, the same tissue sample can be used first for routine HE-staining followed by IHC staining if required, without the need for further sampling of the patient.


  • The staining is highly specific ot the target antigen, depending somewhat on the type of antibody used. Monoclonal antibodies are likely to give a more specific result than polyclonal ones.

Technique (intrinsic) limitations

  • False negative results: eg if an infectious agent is present in very low numbers within a tissue sample, then it simply may not be present within the particular section examined. Neoplastic cell populations may sometimes lose expression of the expected cell markers during the process of malignant transformation.

Technician (extrinsic) limitations

  • Non-specific labeling may obscure the specific staining if severe.
  • Non-specific labeling may confuse an inexperienced observer.

Further Reading


Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Berlato D et al(2012) Evaluation of minichromosome maintenance protein 7 as a prognostic marker in canine cutaneous mass cell tumours. Vet Comp Oncol 10 (2), 125-142 PubMed.
  • Giantin M et al (2012) c-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumours: correlations with post-surgical prognosis. J Vet Diag Invest 24 (1), 116-126 PubMed.
  • Bergin I L et al (2011) Prognostic evaluation of Ki67 threshold value in canine oral melanoma. Vet Pathol 48 (1), 41-53 PubMed.
  • Marconato L, Stefanello D, Valenti P, Bonfanti U et al (2011) Predictors of long-term survival in dogs with high-grade multicentric lymphoma. JAVMA 238 (4), 480-485 PubMed.
  • Maglennon G A et al(2008) Association of Ki67 index with prognosis for intermediate-grade canine cutaneous mast cell tumours. Vet Comp Oncol 6(4), 268-274.
  • Webster J D et al (2008) Evaluation of prognostic markers for canine mast cell tumours treated with vinblastine and prednisone. BMC Vet Res 4, 32 PubMed.
  • Webster J D et al (2008) Cellular proliferation in canine cutaneous mast cell tumours: associations with c-KIT and its role in prognostication. Vet Pathol 44 (3), 98-308 PubMed.
  • Scase T J et al (2006) Canine mast cell tumours: correlation of apoptosis and proliferation markers with prognosis. JVIM 20, 151-158 PubMed.
  • Kiupel M et al(2004) The use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors. Vet Pathol 42(4), 371-377 PubMed.
  • Fournel-Fleury C, Ponce F, Felman P et al (2002) Canine T-cell lymphomas: a morphological, immunological, and clinical study of 46 new cases. Vet Pathol 39, 92-109.
  • Koutinas A F, Polizopoulou S, Baumgaertner W, Lekkas S & Kontos V (2002) Relation of clinical signs to pathological changes in 19 cases of canine distemper encephalomyelitis. J Comp Pathol 126, 47-56.
  • Dobson J M et al (2001) Prognostic variables in canine multicentric lymphosarcoma. JSAP 42 (8), 377-384 PubMed.
  • German A J, Hall E J & Day M J (2001) Characterization of immune cell populations within the duodenal mucosa of dogs with enteropathies. JVIM 15, 14-25.
  • Waly N, Gruffydd-Jones T J, Stokes C R & Day M J (2001) The distribution of leucocyte subsets in the small intestine of normal cats. J Comp Pathol 124, 172-182.
  • German A J, Hall E J, Moore P F, Ringler D J, Newman W & Day M J (1999) Analysis of the distribution of lymphocytes expressing ab and gd T cell receptors and expression of mucosal addressin cell adhesion molecule-1 in the canine intestine.  J CompPathol 121, 249-263.
  • Day M J, Hanlon L & Powell L M (1993) Immune-mediated skin disease in the dog and catJ Comp Pathol 109, 395-407.
  • Zipfel W et al (1992) Demonstration of immunoglobulins and complement in canine and feline autoimmune and non-autoimmune skin diseases with the direct immunofluorescence and indirect immunoperoxidase method. ZentralblVeterinarmed A 39, 494-501.
  • Bradley G A & Mays M B (1990) Immunoperoxidase staining for the detection of autoantibodies in canine autoimmune skin disease; comparison to immunofluorescence results. Vet Immunol Immunopathol 26,105-13.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, UK.

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