Immunocytochemistry in Histology and Cytology

  • Pranab Dey


Immunohistochemistry (IHC) helps to visualize the recognition of the antigen in the tissue with the help of corresponding antibody. The present chapter elucidates the principle of IHC and detailed description of the different types of antigen-antibody detection methods. The relative advantages and disadvantages of each technique have been described. The chapter also covers the methods of collection, fixation and the different techniques of antigen retrieval. Antigen retrieval is possible by microwave oven, pressure cooker heating and water bath heating methods. The steps of routine immunocytochemistry along with the troubleshooting of the techniques are also discussed in detail.


Immunohistochemistry Immunocytochemistry Antigen Antibody Hybridoma technique Monoclonal antibody Polyclonal antibody Affinity Avidity Peroxidase-antiperoxidase method Avidin and biotin method Biotin-streptavidin method Alkaline phosphatase-antialkaline phosphatase method Polymer-based labelling method Catalysed signal amplification Tyramine signal amplification Antigen retrieval Microwave retrieval Immunohistochemistry protocol Troubleshooting in immunocytochemistry 


  1. 1.
    Coons AH, Creech HJ, Jones RN. Immunological properties of an antibody containing a fluorescent group. Exp Biol Med. 1941;47(2):200.CrossRefGoogle Scholar
  2. 2.
    Nakane PK, Pierce GB. Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol. 1967;33:307–18.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J Clin Pathol. 1974;27:14–20.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    McMichael AJ, Pilch JR, Galfre G, Mason DY, Fabre JW, Milstein C. A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody. Eur J Immunol. 1979;9:205–10.CrossRefPubMedGoogle Scholar
  5. 5.
    Erber WN, McLachlan J. Use of APAAP technique on paraffin wax embedded bone marrow trephines. J Clin Pathol. 1989;42:1201.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic label of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J Histochem Cytochem. 1984;32:219.CrossRefPubMedGoogle Scholar
  7. 7.
    Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, Pieri F, Fraternali-Orcioni G, Pileri SA. The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol. 1998;51(7):506–11.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Toda Y, Kono K, Abiru H, Kokuryo K, Endo M, Yaegashi H, Fukumoto M. Application of tyramide signal amplification system to immunohistochemistry: a potent method to localize antigens that are not detectable by ordinary method. Pathol Int. 1999;49:479–83.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Staff S, Kujala P, Karhu R, Rökman A, Ilvesaro J, Kares S, Isola J. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives. J Clin Pathol. 2013;66(9):807–10.CrossRefPubMedGoogle Scholar
  10. 10.
    Shi SR, Cote RJ, Taylor CR. Antigen retrieval techniques: current perspectives. J Histochem Cytochem. 2001;49(8):931–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Fraenkel-Conrat H, Brandon BA, Olcott HS. The reaction of formaldehyde with proteins. IV: participation of indole groups. J Biol Chem. 1947;168:99.PubMedGoogle Scholar
  12. 12.
    Boon ME, Kok LP, Suurmeijer AJH. The MIB-1 method for fine-tuning diagnoses in cervical cytology. In: Shi S-R, Gu J, Taylor CR, editors. Antigen retrieval techniques: immunohistochemistry and molecular morphology. Natick: Eaton; 2000. p. 57–70.Google Scholar
  13. 13.
    Stirling JW. Antigen retrieval and unmasking for immunoelectron microscopy. In: Shi S-R, Gu J, Taylor CR, editors. Antigen retrieval techniques: immunohistochemistry and molecular morphology. Natick: Eaton; 2000. p. 93–113.Google Scholar
  14. 14.
    Shi S-R, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39:741.CrossRefPubMedGoogle Scholar
  15. 15.
    Delahaye M, van der Ham F, van der Kwast TH. Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions. Diagn Cytopathol. 1997;17:115–20.CrossRefPubMedGoogle Scholar
  16. 16.
    Yaziji H, Battifora H, Barry TS, et al. Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: identification of a three-antibody immunohistochemical panel with maximal sensitivity and specificity. Mod Pathol. 2006;19:514–23.CrossRefPubMedGoogle Scholar
  17. 17.
    Chu PG, Weiss LM. Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod Pathol. 2002;15:6–10.CrossRefPubMedGoogle Scholar
  18. 18.
    Busam KJ, Iversen K, Coplan KA, Old LJ, Stockert E, Chen YT, McGregor D, Jungbluth A. Immunoreactivity for A103, and antibody to melan-A (Mart-1), in adrenocortical and other steroid tumors. Am J Surg Pathol. 1998;22:57–63.CrossRefPubMedGoogle Scholar
  19. 19.
    Bodey B, Bodey B Jr, Kaiser HE. Immunocytochemical detection of prostate specific antigen expression in human breast carcinoma cells. Anticancer Res. 1997;17:2577–81.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Saleem TB, Ahmed I. Gastrointestinal stromal tumour--evolving concepts. Surgeon. 2009;7(1):36–41.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Pranab Dey
    • 1
  1. 1.Education and Research (PGIMER)Post Graduate Institute of Medical Education and Research (PGIMER)ChandigarhIndia

Personalised recommendations