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Originally published as JHC exPRESS on April 13, 2009.
doi:10.1369/jhc.2009.953612
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Journal of Histochemistry and Cytochemistry
Volume 57 (10): 899-905, 2009
Copyright ©The Histochemical Society, Inc.

SIMPLE: A Sequential Immunoperoxidase Labeling and Erasing Method

George Glass, Jason A. Papin and James W. Mandell

Department of Biomedical Engineering, (GG,JAP) and Department of Pathology (Neuropathology) (JWM), University of Virginia, Charlottesville, Virginia

Correspondence to: James W. Mandell, University of Virginia, Box 800904, UVa Health System, Charlottesville, VA 22908. E-mail: jwm2m{at}virginia.edu

The ability to simultaneously visualize expression of multiple antigens in cells and tissues can provide powerful insights into cellular and organismal biology. However, standard methods are limited to the use of just two or three simultaneous probes and have not been widely adopted for routine use in paraffin-embedded tissue. We have developed a novel approach called sequential immunoperoxidase labeling and erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with a rapid non-destructive method for antibody–antigen dissociation, we demonstrate the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. SIMPLE is greatly facilitated by the use of a whole-slide scanner, which can capture the results of each sequential stain without any information loss. (J Histochem Cytochem 57:899–905, 2009)

Key Words: immunohistochemistry • colocalization • multiple • antigens


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