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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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
Shi S, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49(8):931–937
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
Huang SN, Minassian H, More JD (1976) Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Investig 35(4):383–390
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
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
Pileri SA, Roncador G, Ceccarelli C et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183(1):116–123
McNicol R (1998) Optimizing immunohistochemistry: antigen retrieval and signal amplification. Histopathology 32(2):97–103
Werner M, Wasielewski RV, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105(4):253–260
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
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
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
Bratthauer GL (2010) The avidin-biotin complex (ABC) method and other avidin-biotin binding methods. Methods Mol Biol 588:257–270
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
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
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
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
Faget L, Hnasko TS (2015) Tyramide signal amplification for immunofluorescent enhancement. Methods Mol Biol 1318:161–172
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
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