Immunohistochemical Detection of Tissue and Cellular Antigens


Antibodies are excellent tools for detecting specific molecules in cells and tissues. Techniques have been developed utilising antibodies to visualise otherwise invisible, and indistinguishable, cells, subcellular structures and molecules. Collectively, these techniques are termed immunohistochemistry, which is used extensively in many fields from research to clinical pathology. A plethora of antibodies and visualisation systems, generally of increasing quality, has been launched on the market allowing for an ever‐increasing technical sensitivity and specificity. This development, however, also challenges the users owing to varying or even incompatible results reported in the literature. In the era of personalised medicine, correct identification of protein markers for targeted treatment in many types of cancer requires meticulous laboratory handling and standardised reading. Internal quality control and external quality assessment have become increasingly important.

Key Concepts

  • Immunohistochemistry is technically complex, and no aspect of this complexity can be ignored, from the moment of collecting the specimen to issuance of the final report.
  • Preanalytical steps include proper sampling, fixation and processing of tissues and cells.
  • Analytical steps include selection of specific and avid antibodies and a carefully calibrated protocol.
  • The large majority of epitopes in formalin fixed tissue can only be sufficiently detected by heat‐induced epitope retrieval in an alkaline buffer.
  • Selection and on‐slide mounting of appropriate control tissues are mandatory.
  • Postanalytical steps include identification of a good signal‐to‐noise ratio and correct subcellular localisation of the chromogene.
  • Assessment of the staining reaction and interpretation of the staining result must be performed in a proper context, for example, with the literature and the clinical setting.

Keywords: immunohistochemistry; immunocytochemistry; immunohistology; microscopy; quality assurance

Figure 1. Flow diagram of essential processing steps before the immunostaining procedure. For explanation, see text.
Figure 2. Schematic representation of the most common immunohistochemical staining procedures. The steps for each procedure are numbered. For explanation, see text and Table.
Figure 3. Immunoperoxidase staining of a rat spleen section. An acetone‐fixed frozen section from rat spleen was incubated first with a mouse monoclonal antibody directed to B cells (HIS14) and followed by a horseradish peroxidase‐labelled secondary antibody directed against mouse antibodies. The peroxidase label was revealed using diaminobenzidine, resulting in a brown precipitated reaction product at the site of binding of the monoclonal antibody to the cells. B cells in this section are stained brown and are located in the typical B cell areas: follicles (F) and marginal zone (MZ). T lymphocytes are located in the periarteriolar lymphocyte sheath (PALS) and are unstained.
Figure 4. Two‐colour immunohistochemical staining of heart tissue after transplantation. To identify infiltrating T and B cells in cardiac tissue undergoing chronic rejection in rats, an indirect double‐staining technique was performed. Fixed cryostat section of transplanted rat heart was incubated with a mixture of an IgG1 mouse monoclonal antibody directed against rat T cells (OX19) and an IgG2b mouse monoclonal antibody directed against rat B cells (HIS24). These antibodies were revealed with alkaline phosphatase‐conjugated goat antimouse IgG1 and horseradish peroxidase‐conjugated goat antimouse IgG2b, respectively. In this section, the infiltrating T cells are stained blue and B cells are stained red. Courtesy of J. L. Hillebrands, University of Groningen.
Figure 5. Immunoperoxidase and immunofluorescence staining of human skin. Frozen sections of human skin were stained for laminin 5 using a mouse monoclonal antibody to laminin 5, followed by polyclonal rabbit antimouse antibodies, conjugated to horseradish peroxidase (a) or to FITC (b). Peroxidase was revealed using diaminobenzidine (brown) and nuclei were stained with haematoxylin (blue). Laminin 5 is expressed by the epidermal basement membrane zone (arrow). Ep, epidermis. Courtesy of M. van der Deen and Dr. M. C. J. M. de Jong, University of Groningen.
Figure 6. Immunofluorescence staining of human skin. Human skin sections were stained by immunofluorescence as described for Figure, using monoclonal antibodies directed to bullous pemphigoid antigen BP230 (a) or BP180ic (b). Nuclei are stained blue using bisbenzimide. BP230 is expressed by the epidermal basement membrane zone (arrow) and BP180ic also by the lamina basalis of the epidermis (Ep). Courtesy of M. van der Deen and Dr. M. C. J. M. de Jong, University of Groningen.
Figure 7. Three‐colour immunofluorescence staining of mouse lymphocytes after BrdU incorporation. These photographs show two peritoneal B cells (B‐1 cells) of a mouse fed with BrdU in the drinking water. Peritoneal cavity cells were stained with a fluoresceinated (FITC, green fluorescence) rat monoclonal antibody directed CD5 (b) and a rhodamine (TRITC, red fluorescence) conjugated rat monoclonal antibody to mouse IgM (c). After cytocentrifugation, fixation and denaturation of the cells, BrdU incorporation was visualised with an IgG1 mouse monoclonal antibody to BrdU, followed by biotinylated goat antimouse IgG1 and finally AMCA (blue fluorescence)‐conjugated avidin (d). The pictures clearly show that both cells express CD5 and IgM, whereas only one of them had incorporated BrdU and thus had gone through a cell cycle during the period of BrdU feeding. (a) Phase contrast picture of the two peritoneal cells. Reproduced with permission from Deenen and Kroese (1993) © John Wiley and Sons Ltd.
Figure 8. Confocal scanning laser microscopy image of six optical planes of a hepatocyte couplet. Some hepatocytes isolated from a (rat) liver stay attached to each other, forming a bile canaliculus (apical domain) in culture. Apical trafficking of the multidrug resistance protein 2 (MRP2), a glutathione S‐conjugate transporter, was studied in hepatocyte couplets. The image shows six optical planes through a hepatocyte couplet that was treated with an inhibitor of microtubule polymerisation (nocodazole) and then stained for MRP2 with a polyclonal antibody followed by a FITC‐labelled secondary antibody. The upper left image shows the top of the couplet and the lower right image shows the bottom (the side that is attached to the culture dish). MRP2 is predominantly localised in a vesicular compartment just below the plasma membrane, indicating that apical trafficking of MRP2 is dependent on microtubules. Courtesy of Dr. J. Roelofsen, University of Groningen.
Figure 9. Immunoelectronmicroscopy of brain tissue using immunogold. Ultrathin sections of rabbit brain were stained for the presence of the most frequently occurring inhibitory neurotransmitter GABA using a postembedding, indirect immunogold method. Sections were incubated with polyclonal rabbit anti‐GABA antibody, followed by secondary antibodies directed against rabbit antibodies conjugated with 15 nm gold particles. The gold particles, and thus GABA, are predominantly observed in axonal terminal (AT) in contact with a proximal dendrite (D) of a neuron in the nucleus of the optic tract in the rabbit. Note that mitochondria (arrow) within the labelled terminal also exhibit significant amounts of GABA. Courtesy of Dr. J. J. L. van der Want, University of Groningen.


Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. Journal of Histochemistry and Cytochemistry 40: 1457–1463.

Bobrow MN, Harris TD, Shaugnessy KJ and Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. Journal of Immunological Methods 125: 279–285.

Chilosi M, Lestani M, Pedron S, et al. (1994) A rapid immunostaining method for frozen sections. Biotechnic and Histochemistry 69: 235–239.

Coons AH, Creech HJ and Jones RN (1941) Immunologic properties of an antibody containing a fluorescent group. Proceedings of the Society for Experimental Biology 47: 200–202.

Deenen GJ and Kroese FGM (1993) Kinetics of peritoneal B‐1A cells (CD5 B‐cells) in young‐adult mice. European Journal of Immunology 23 (1): 12–16.

Emmert‐Buck MR, Bonner RF, Smith PD, et al. (1996) Laser capture microdissection. Science 274: 998–1001.

Gavrielli Y, Sherman Y and Ben‐Sasson SA (1992) Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. Journal of Cell Biology 119: 493–501.

Guesdon JL, Ternynck T and Avrameas S (1979) The use of avidin–biotin interaction in immunoenzymatic techniques. Journal of Histochemistry and Cytochemistry 27: 1131–1139.

Hsu SM, Raine L and Fanger H (1981) Use of avidin‐biotin‐peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled PAP procedures. Journal of Histochemistry and Cytochemistry 33: 219–229.

Kohler G and Milstein C (1975) Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256: 495–497.

Küppers R, Zhao M, Hansmann ML and Rajewsky K (1993) Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO Journal 12: 4955–4967.

Shotton DM (1989) Confocal scanning microscopy and its applications for biological specimens. Journal of Cell Science 95: 175–206.

Sternberger LA, Hardy JPH, Cuculis JJ and Meyer GH (1970) The unlabeled antibody–enzyme method of immunochemistry. Preparation of soluble antigen–antibody complex (horseradish peroxidase anti‐horseradish peroxidase) and its use in identification of spirochetes. Journal of Histochemistry and Cytochemistry 33: 577–580.

Vyberg M and Nielsen S (1998) Dextran polymer conjugate two‐step visualization system for immunohistochemistry. A comparison of EnVision + with two three step avidin–biotin techniques. Applied Immunohistochemistry 6: 3–10.

Vyberg M and Nielsen S (2016) Proficiency testing in immunohistochemistry – experiences from Nordic Immunohistochemical Quality Control (NordiQC). Virchows Archiv 468: 19–29.

Further Reading

Bancroft JD and Cook HC (1994) Manual of Histological Techniques and Their Diagnostic Applications. Edinburgh, UK: Churchill Livingstone.

Beesley JE (ed) (1993) Immunocytochemistry: A Practical Approach. Oxford, UK: Oxford University Press.

Bullock G and Petrusz P (eds) (1986–1989) Techniques in Immunocytochemistry, vols 1–4. San Diego, CA: Academic Press.

Hayat M (ed) (1990) Colloidal Gold: Principles, Methods and Applications. San Diego, CA: Academic Press.

Kroese FGM (1997) Immunohistochemistry and immunopathology. In: Herzenberg LA, Herzenberg DW and Weir C (eds) Weir's Handbook of Experimental Immunology, 5th edn, vol. IV, pp. 203.1–210.8. Cambridge, UK: Blackwell Science.

Pearse A (1985) Histochemistry, Theoretical and Applied. Edinburgh, UK: Churchill Livingstone.

Polak JM and Van Noorden S (1997) Introduction to Immunocytochemistry, 2nd edn. Oxford, UK: BIOS Scientific.

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Vyberg, Mogens, and Nielsen, Søren(Jun 2017) Immunohistochemical Detection of Tissue and Cellular Antigens. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001176.pub2]