Equis ISSN 2398-2977

Immunostaining: immunohistochemistry / immunocytochemistry - direct and indirect

Synonym(s): Immunofluorescence

Contributor(s): Alastair Foote, Vetstream Ltd


  • Immunostaining is used to detect a specific target antigen within a tissue biopsy (immunohistochemistry) or associated with individual cells on a cytological (smear or cytospin) preparation (immunocytochemistry). 
  • The target antigen is detected by an antibody, and the binding of the antigen-antibody is demonstrated by an indicator system. 
  • The indicator may be the emission of fluorescence when exposed to light of a particular wavelength (immunofluorescence), or deposition of color at the site of antibody binding following an enzyme-substrate reaction. 
  • With direct immunofluorescence, the indicator (fluorochrome) is bound directly to the primary antibody. Indirect immunofluorescence/immunohistochemistry utilizes a secondary antibody that recognizes the primary antibody and is then demonstrated by an indicator system (fluorescent or histochemical).
  • The direct method requires fewer steps and is therefore more rapid. It also avoids problems of non-specific binding/cross-reactivity that can occur with the secondary antibody in indirect methods. However, is it less sensitive since there is no amplification of the signal and is used less commonly than the indirect method. 
  • The use of an indirect technique increases the sensitivity of the method due to signal amplification (several secondary antibodies can bind to different antigenic sites on the primary antibody). A range of other such amplification techniques are available, eg the detecting antibody may be conjugated to biotin, which in turn binds to the avidin-linked enzyme. The indirect method also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies need to be generated.


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  • For formalin-fixed tissue, pre-treatment of sections is required to remove wax from the tissue. 
  • Fixed tissue sections are usually pre-treated to open the tissue and expose antigenic sites to increase the likelihood of the detecting antibody being able to bind to the relevant antigen (antigen retrieval). 
  • A range of such pre-treatment techniques are utilized, eg incubation in a trypsin or pronase solution, or incubation in citrate buffer with microwaving. 
  • Cytological preparations and frozen tissue sections may be fixed in cold acetone before immunolabeling. 
  • Following pre-treatment, the sections are then incubated with the detecting antibody. This occurs for variable time periods (from 30 min to 16 h) and temperatures, depending upon the nature of the antibody. 
  • In simple direct immunolabeling, the fluorochrome or enzyme are conjugated to the primary antibody. 
  • Where a secondary antibody is used (indirect immunolabeling), a subsequent period of incubation is required after thorough washing of the tissue to remove unbound primary antibody. 
  • The use of a secondary antibody necessitates a blocking step to reduce non-specific background staining.  This is performed by incubating the sections with dilute serum of the animal species in which the secondary antibody was raised. 
  • For immunofluorescence, the section is then coverslipped under an aqueous mountant and examined with a fluorescence microscope. Fluorescence will be localized to the area of antibody deposition. 
  • A range of fluorochromes are available that emit light at different wavelengths, but that most commonly used (fluorescein isothiocyanate or FITC) produces an apple-green fluorescence. 
  • It is possible to probe sections for several different antigens using several different antibodies coupled to different fluorochromes, and thus build up a composite picture. 
  • For immunohistochemistry, the final stage of the reaction involves incubation of the tissue with chromogenic substrate. 
  • The most commonly used enzyme is peroxidase, and the most commonly used substrate-chromagen is peroxide with diaminobenzidine. This reaction produces a chocolate-brown deposit at the site of antibody binding within the tissue. 
  • The section can be counterstained with haematoxylin to provide an indication of tissue morphology and, after mounting under DPX, can be examined by standard light microscopy.


  • The laboratory should include appropriate positive and negative controls in each test.


  • Not all laboratories will offer immunolabeling, but most will be able to refer material on to other laboratories.



  • The direct immunolabeling method is a relatively sensitive technique, eg organisms that are not obvious in routine H&E stained tissue sections may be identified by immunohistochemistry. 
  • The sensitivity of the test can be increased by the use of amplification techniques such as the sandwich of two antibodies, or the biotin-avidin method.


  • The specificity will be related to the specificity of the detecting antibody. 
  • In general, monoclonal antibodies are likely to give a more specific result than polyclonal antisera. 
  • An antiserum known to cross-react with multiple species of microbe, will still have this limitation when applied to an immunohistochemical technique.

Predictive value

  • The success of the technique will have a number of limitations. 
  • A higher success rate may be obtained by screening multiple biopsies or cytological preparations, by sampling from appropriate sites, by the use of particular antibodies that are optimal for use in this technique, and by the experience of the observer who interprets the test.

Technique (intrinsic) limitations

  • Immunolabeling is a subjective test, the result of which depends on the interpretation made by the observer. 
  • A descriptive or yes/no answer may be given. 
  • Precision can be increased by requesting that the labeling is enumerated, eg the percentage of positively labeled cells within a tumor population may be determined to give a more objective result.

Result Data

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Further Reading


Refereed papers

  • Recent references from PubMed and VetMedResource
  • Clift S J & Penrith M L (2010) Tissue and cell tropism of African horse sickness virus demonstrated by immunoperoxidase labeling in natural and experimental infection in horses in South Africa. Vet Pathol 47 (4), 690-697 PubMed
  • de Bruijn C M, Veenman J N, Rutten V P, Teske E, van Nieuwstadt R A & van den Ingh T S (2007) Clinical, histopathological and immunophenotypical findings in five horses with cutaneous malignant lymphoma. Res Vet Sci 83 (1), 63-72 PubMed
  • Ramos-Vara J A (2005) Technical aspects of immunohistochemistry. Vet Pathol 42 (4), 405-426 PubMed.
  • Gerst S, Borchers K, Gower S M & Smith K C (2003) Detection of EHV-1 and EHV-4 in placental sections of naturally occurring EHV-1- and EHV-4-related abortions in the UK: use of the placenta in diagnosis.  Equine Vet J 35 (5), 430-433 PubMed.