FLUORESCEIN-LABELED TYRAMIDE STRONGLY ENHANCES THE DETECTION OF LOW BROMODEOXYURIDINE INCORPORATION LEVELS

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an ampl ification method for weak BrdU immunosignals. The tyramide signal ampl ification method based on catalyzed reporter deposition (CARD) uses fl uorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radical s 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 feta l bovine serum supplemented by growth factors. Under these culture con ditions, the BrdU immunosignal was hard to detect but could be enhance d 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 CARD BrdU lab eling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might al so prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.