Skip to main content
SpringerLink
Log in
Book cover

Success in Academic Surgery: Basic Science pp 137–155Cite as

Considerations for Immunohistochemistry

  • Chapter
  • First Online:

  • 1247 Accesses

Part of the book series: Success in Academic Surgery ((SIAS))

Abstract

Immunohistochemistry (IHC) is defined as the localization of specific antigens in tissues by staining with antibodies labeled with fluorescent or pigmented material. It combines anatomical, immunological, and biochemical techniques to identify discrete tissue components. IHC is a critical research tool for investigations involving animal or human tissues. It relies on the ability of antibodies to recognize and bind antigens with high specificity. It can be used to identify specific tissue components, cells, and cell processes, such as cell proliferation, apoptosis, and oxidative stress. Like any research technique, obtaining optimal results using IHC requires the appropriate controls, as well as individual adjustments in technique. In the past, this process was semiquantitative at best; however, new advances are making it possible to obtain quantitative results using IHC. This chapter serves to review the applications of IHC, basic principles, techniques, troubleshooting, and analysis of IHC.

This is a preview of subscription content, log in via an institution.

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   69.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

References

  1. Yu CC, Woods AL, Levison DA. The assessment of cellular proliferation by immunohistochemistry: a review of currently available methods and their applications. Histochem J. 1992;24:121–31.

    Article  PubMed  CAS  Google Scholar 

  2. Cheuk W, Chan JK. Subcellular localization of immunohistochemical signals: knowledge of the ultrastructural or biologic features of the antigens helps predict the signal localization and proper interpretation of immunostains. Int J Surg Pathol. 2004;12:185–206.

    Article  PubMed  CAS  Google Scholar 

  3. Poynter ME, Janssen-Heininger YM, Buder-Hoffmann S, Taatjes DJ, Mossman BT. Measurement of oxidant-induced signal transduction proteins using cell imaging. Free Radic Biol Med. 1999;27:1164–72.

    Article  PubMed  CAS  Google Scholar 

  4. Duan WR, Garner DS, Williams SD, et al. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol. 2003;199:221–8.

    Article  PubMed  CAS  Google Scholar 

  5. Holubec H, Payne CM, Bernstein H, et al. Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa. J Histochem Cytochem. 2005;53:229–35.

    Article  PubMed  CAS  Google Scholar 

  6. Shigenaga MK, Aboujaoude EN, Chen Q, Ames BN. Assays of oxidative DNA damage biomarkers 8-oxo-2′-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 1994;234:16–33.

    Article  PubMed  CAS  Google Scholar 

  7. Raina AK, Perry G, Nunomura A, Sayre LM, Smith MA. Histochemical and immunocytochemical approaches to the study of oxidative stress. Clin Chem Lab Med. 2000;38:93–7.

    Article  PubMed  CAS  Google Scholar 

  8. Hayes AJ, Hughes CE, Caterson B. Antibodies and immunohistochemistry in extracellular matrix research. Methods. 2008;45:10–21.

    Article  PubMed  CAS  Google Scholar 

  9. Owen GR, Meredith DO, ap Gwynn I, Richards RG. Focal adhesion quantification – a new assay of material biocompatibility? Review. Eur Cell Mater. 2005;9:85–96; discussion 85–96.

    PubMed  CAS  Google Scholar 

  10. Coons AH, Creech HJ, Jones RN. Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med. 1941;47:200–2.

    Article  CAS  Google Scholar 

  11. Avrameas S. Enzyme markers: their linkage with proteins and use in immuno-histochemistry. Histochem J. 1972;4:321–30.

    Article  PubMed  CAS  Google Scholar 

  12. Nakane PK, Pierce Jr GB. Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol. 1967;33:307–18.

    Article  PubMed  CAS  Google Scholar 

  13. Adams JC. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem. 1992;40:1457–63.

    Article  PubMed  CAS  Google Scholar 

  14. Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods. 1989;125:279–85.

    Article  PubMed  CAS  Google Scholar 

  15. Sternberger LA, Hardy Jr PH, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem. 1970;18:315–33.

    Article  PubMed  CAS  Google Scholar 

  16. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:577–80.

    Article  PubMed  CAS  Google Scholar 

  17. Sabattini E, Bisgaard K, Ascani S, et al. 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;51:506–11.

    Article  PubMed  CAS  Google Scholar 

  18. Shi SR, 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;39:741–8.

    Article  PubMed  CAS  Google Scholar 

  19. True LD. Atlas of diagnostic immunohistopathology. Philadelphia: J. B. Lippincott Company; 1990.

    Google Scholar 

  20. Kiernan J. Histological and histochemical methods, theory and practice. Bloxham: Scion Publishing Limited; 2008.

    Google Scholar 

  21. Kiernan J. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today. 2000;00–1:8–12.

    Google Scholar 

  22. Nissanov J, Bertrand L, Tretiak O. Cryosectioning distortion reduction using tape support. Microsc Res Tech. 2001;53:239–40.

    Article  PubMed  CAS  Google Scholar 

  23. Sompuram SR, Vani K, Messana E, Bogen SA. A molecular mechanism of formalin fixation and antigen retrieval. Am J Clin Pathol. 2004;121:190–9.

    Article  PubMed  CAS  Google Scholar 

  24. Boenisch T. Heat-induced antigen retrieval: what are we retrieving? J Histochem Cytochem. 2006;54:961–4.

    Article  PubMed  CAS  Google Scholar 

  25. Dabbs D. Diagnostic immunohistochemistry. Philadelphia: Churchill, Livingstone, Elsevier; 2006.

    Google Scholar 

  26. van Gijlswijk RP, et al. Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem. 1997;45:375–82.

    Article  PubMed  Google Scholar 

  27. Hatanaka Y, Imaoka Y, Torisu K, et al. A simplified, sensitive immunohistochemical detection system employing signal amplification based on fluorescyl-tyramide/antifluorescein antibody reaction: its application to pathologic testing and research. Appl Immunohistochem Mol Morphol. 2008;16:87–93.

    PubMed  CAS  Google Scholar 

  28. Schweitzer B, Wiltshire S, Lambert J, et al. Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci U S A. 2000;97:10113–9.

    Article  PubMed  CAS  Google Scholar 

  29. Wiltshire S, O’Malley S, Lambert J, et al. Detection of multiple allergen-specific IgEs on microarrays by immunoassay with rolling circle amplification. Clin Chem. 2000;46:1990–3.

    PubMed  CAS  Google Scholar 

  30. Sweeney E, Ward TH, Gray N, et al. Quantitative multiplexed quantum dot immunohistochemistry. Biochem Biophys Res Commun. 2008;374:181–6.

    Article  PubMed  CAS  Google Scholar 

  31. Burry RW. Controls for immunocytochemistry: an update. J Histochem Cytochem. 2011;59:6–12.

    Article  PubMed  CAS  Google Scholar 

  32. Ramos-Vara JA. Principles and methods of immunohistochemistry. Methods Mol Biol. 2011;691:83–96.

    Article  PubMed  Google Scholar 

  33. Fetsch PA, Abati A. The clinical immunohistochemistry laboratory: regulations and troubleshooting guidelines. Methods Mol Biol. 2010;588:399–412.

    Article  PubMed  Google Scholar 

  34. Cregger M, Berger AJ, Rimm DL. Immunohistochemistry and quantitative analysis of protein expression. Arch Pathol Lab Med. 2006;130:1026–30.

    PubMed  CAS  Google Scholar 

  35. Taylor CR, Levenson RM. Quantification of immunohistochemistry–issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006;49:411–24.

    Article  PubMed  CAS  Google Scholar 

  36. Kononen J, Bubendorf L, Kallioniemi A, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4:844–7.

    Article  PubMed  CAS  Google Scholar 

  37. Kirkeby S, Thomsen CE. Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software. J Immunol Methods. 2005;301:102–13.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Vascular Surgery, Department of Surgery, VA Puget Sound Health Care System, Seattle, Washington, USA

    Gale L. Tang MD

  2. Department of Surgery, Stanford University, 300 Pasteur Dr., H3640, Stanford, CA, 94305, USA

    Wei Zhou MD

Authors
  1. Gale L. Tang MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Zhou MD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei Zhou MD .

Editor information

Editors and Affiliations

  1. Professor of Surgery, Vice Chair of Research, Edward G. Elcock Professor of Surgical Research, Department of Surgery, Northwestern University, Chicago, IL, USA, and Co-Chief, Peripheral Vascular Surgery, Jesse Brown VA Medical Center, Chicago, Illinois, USA

    Melina R. Kibbe

  2. Division of Cardiothoracic Surgery, Baylor College of Medicine, and Department of Cardiovascular Surgery, Texas Heart Institute, St. Luke's Episcopal Hospital, Houston, Texas, USA

    Scott A. LeMaire

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer-Verlag London

About this chapter

Cite this chapter

Tang, G.L., Zhou, W. (2014). Considerations for Immunohistochemistry. In: Kibbe, M., LeMaire, S. (eds) Success in Academic Surgery: Basic Science. Success in Academic Surgery. Springer, London. https://doi.org/10.1007/978-1-4471-4736-7_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4471-4736-7_10

  • Published:

  • Publisher Name: Springer, London

  • Print ISBN: 978-1-4471-4735-0

  • Online ISBN: 978-1-4471-4736-7

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation