Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels

Abstract

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 dear-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.