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Journal of Histochemistry and Cytochemistry, Vol. 45, 315-320, Copyright © 1997 by The Histochemical Society, Inc.

TECHNICAL NOTE

Fluorescein-labeled Tyramide Strongly Enhances the Detection of Low Bromodeoxyuridine Incorporation Levels

Jimmy Van heusdena, Paul de Jongd, Frans Ramaekersa, Hélène Bruwiereb, Marcel Borgersc, and Gerda Smetsb
a Department of Molecular Cell Biology and Genetics, University of Limburg, Maastricht, The Netherlands
b Department of Oncology, Janssen Research Foundation, Beerse, Belgium
c Department of Morphology, Janssen Research Foundation, Beerse, Belgium
d DuPont NEN, Life Science Products, Dordrecht, The Netherlands

Correspondence to: Jimmy Van heusden, Janssen Research Foundation, Dept of Oncology, Turnhoutseweg 30, B-2340 Beerse, Belgium.

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses. (J Histochem Cytochem 45:315-319, 1997)

Key Words: BrdU, CARD, immunocytochemistry, MCF-7, signal amplification, tyramide


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