Skip to main content

Antigen Retrieval and Signal Amplification

Part of the Methods in Molecular Biology book series (MIMB,volume 2422)

Abstract

Immunohistochemistry (IHC) offers a valuable method for determining the spatial distribution of proteins in cells and tissues. Fixation of tissues prior IHC enables their long-term stability and preserves tissue morphology; however, downstream analysis of protein localization within fixed samples can be complicated by cross-links formed between proteins during formalin fixation which mask target epitopes. Antigen Retrieval (AR) is a process introduced to reverse such cross-links, improving the sensitivity of antibody-based protein detection, and can be performed using protease- or heat-based approaches. Even following AR, low abundance target proteins may require additional amplification for sensitive visualization. The development of amplification approaches such as the use of biotinylated secondary antibodies with avidin–biotin complex and tyramide signal amplification greatly improve the sensitivity of IHC, enabling a wider range of epitopes to be detected when coupled with AR.

Key words

  • Antigen retrieval
  • Heat-induced epitope retrieval
  • Enzymatic
  • Proteases
  • Signal amplification

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-1948-3_5
  • Chapter length: 10 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   139.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-1948-3
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Hardcover Book
USD   179.99
Price excludes VAT (USA)

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bogen SA, Vani K, Sompuram SR (2009) Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced crosslinks. Biotech Histochem 84(5):207–215

    CAS  CrossRef  Google Scholar 

  2. Shi S, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49(8):931–937

    CAS  CrossRef  Google Scholar 

  3. Miller RT, Swanson PE, Wick MR (2000) Fixation and epitope retrieval in diagnostic immunohistochemistry: a concise review with practical considerations. Appl Immunohistochem Mol Morphol 8(3):228–235

    CAS  CrossRef  Google Scholar 

  4. Huang SN, Minassian H, More JD (1976) Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Investig 35(4):383–390

    CAS  PubMed  Google Scholar 

  5. D’Amico F, Skarmoutsou E, Stivala F (2009) State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods 341(1–2):1–18

    CrossRef  Google Scholar 

  6. Shi SR, Key ME, Kalra KL (1991) 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 39(6):741–748

    CAS  CrossRef  Google Scholar 

  7. Pileri SA, Roncador G, Ceccarelli C et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183(1):116–123

    CAS  CrossRef  Google Scholar 

  8. McNicol R (1998) Optimizing immunohistochemistry: antigen retrieval and signal amplification. Histopathology 32(2):97–103

    CAS  CrossRef  Google Scholar 

  9. Werner M, Wasielewski RV, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105(4):253–260

    CAS  CrossRef  Google Scholar 

  10. Krenacs L, Krenacs T, Stelkovics E, Raffeld M (2010) Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Methods Mol Biol 588:103–119

    CAS  CrossRef  Google Scholar 

  11. Shi S, Shi Y, Taylor CR (2011) Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J Histochem Cytochem 59(1):13–32

    CAS  CrossRef  Google Scholar 

  12. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29(4):577–580

    CAS  CrossRef  Google Scholar 

  13. Bratthauer GL (2010) The avidin-biotin complex (ABC) method and other avidin-biotin binding methods. Methods Mol Biol 588:257–270

    CAS  CrossRef  Google Scholar 

  14. Elias JM, Margiotta M, Gaborc D (1989) Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), Avidin–biotin peroxidase complex (ABC), and peroxidase-labeled Avidin–biotin (LAB) methods. Am J Clin Pathol 92(1):62–67

    CAS  CrossRef  Google Scholar 

  15. Toda Y, Kono K, Abiru H et al (1999) Application of tyramide signal amplification system to immunohistochemistry: a potent method to localize antigens that are not detectable by ordinary method. Pathol Int 49(5):479–483

    CAS  CrossRef  Google Scholar 

  16. Tóth ZE, Mezey E (2007) Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J Histochem Cytochem 55(6):545–554

    CrossRef  Google Scholar 

  17. Stack EC, Wang C, Roman KA, Hoyt CC (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70(1):46–58

    CAS  CrossRef  Google Scholar 

  18. Faget L, Hnasko TS (2015) Tyramide signal amplification for immunofluorescent enhancement. Methods Mol Biol 1318:161–172

    CrossRef  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Luis Del Valle .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Dunkenberger, L., Del Valle, L. (2022). Antigen Retrieval and Signal Amplification. In: Del Valle, L. (eds) Immunohistochemistry and Immunocytochemistry. Methods in Molecular Biology, vol 2422. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1948-3_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1948-3_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1947-6

  • Online ISBN: 978-1-0716-1948-3

  • eBook Packages: Springer Protocols