Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry

Laboratory Investigation; a Journal of Technical Methods and Pathology
Arlin B RogersJames G Fox

Abstract

Nonspecific antibody binding is the primary source of confounding background in immunohistochemistry (IHC). Based on observed patterns of background staining, and the known spontaneous reduction of immunoglobulin disulfide bonds in vivo and in vitro, we tested the hypothesis that nonspecific antibody binding in IHC is mediated by sulfhydryl interactions. Coincubation of primary antibodies with reduced glutathione (GSH), L-cysteine, iodoacetic acid, Ellman's reagent and other thiophilic reagents in pH 8 tris-EDTA (TE) buffer inhibited background staining. In contrast, oxidized glutathione (GSSG) exerted no effect. When empirically optimized, coincubation of GSH with primary antibodies significantly improved IHC signal:noise ratio. Tissue preincubation with mercaptans, soft and borderline metals, and other sulfhydryl-reactive reagents also inhibited background staining, but at the expense of target sensitivity. ELISA results confirmed direct binding between murine serum antibodies and GSH in a nonantigen-dependent manner. In summary, thiol-reactive compounds prevent nonspecific antibody binding in IHC. We propose a mechanism whereby nonspecific background resulting from formation of disulfide bridges and other sulfhydryl bonds be...Continue Reading

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Citations

Jan 24, 2009·Proceedings of the National Academy of Sciences of the United States of America·S E ErdmanJ G Fox
Aug 20, 2008·Infection and Immunity·Megan E McBeeDavid B Schauer
Dec 24, 2009·Toxicologic Pathology·J T PainterR A Herbert
Mar 18, 2011·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Allan WangJoanna L Jankowsky
Feb 23, 2012·Scientific Reports·Igor BuchwalowMarkus Tiemann
Jan 25, 2014·Veterinary Pathology·J PangJ G Fox
Nov 11, 2014·Helicobacter·Zeli ShenJames G Fox
Aug 1, 2013·Science and Technology of Advanced Materials·Shao-Hsuan ChuagChien-Fu Chen
Oct 5, 2011·The American Journal of Pathology·Ian N HinesArlin B Rogers

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