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Journal of Histochemistry and Cytochemistry, Vol. 47, 421-430, April 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Immunogold Signal Amplification: Application of the CARD Approach to Electron Microscopy

Gaétan Mayera and Moïse Bendayana
a Department of Pathology and Cell Biology, Université de Montréal, Montreal, Quebec, Canada

Correspondence to: Moïse Bendayan, Dept. of Pathology and Cell Biology, Université de Montréal, CP 6128 Succ. Centre Ville, Montreal, Quebec H3C 3J7, Canada.

Catalyzed reporter deposition (CARD) is a technique that allows amplification of routine immunolabeling in light microscopy. This procedure takes advantage of the horseradish peroxidase (HRP) from an HRP–avidin complex to catalyze the accumulation of reporter-conjugated tyramine (a phenolic compound) onto a surface displaying biotinylated antigen–antibody complexes. The large amount of labeled tyramine deposited allows the detection of an antigenic site with multiple reporter molecules. In this study we modified this amplification protocol to combine it with the immunogold technique for the ultrastructural localization of antigens in electron microscopy. We constructed various tyramide conjugates that permit the combination of this amplification method with a particulate colloidal gold marker. The new probes yield results of high specificity and enhanced intensity. Assessment of the level of resolution of the labeling has demonstrated that, in spite of the amplification, the resolution remains very good. Therefore, once associated, the immunogold and the CARD techniques lead to specific, high-resolution, sensitive and amplified signals that exhibit the advantages of both approaches. (J Histochem Cytochem 47:421–429, 1999)

Key Words: catalyzed reporter deposition, (CARD), signal amplification, colloidal gold immunocytochemistry, tyramide conjugates, electron microscopy


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