THE USE OF PEROXIDASE-MEDIATED DEPOSITION OF BIOTIN-TYRAMIDE IN COMBINATION WITH TIME-RESOLVED FLUORESCENCE IMAGING OF EUROPIUM CHELATE LABEL IN IMMUNOHISTOCHEMISTRY AND IN-SITU HYBRIDIZATION

The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quant um yield. To increase the intensity of the delayed fluorescence, we ha ve used a recently introduced peroxidase-mediated amplification system . This system results in a large accumulation of biotin-tyramide, whic h is detected using streptavidin-europium chelate as label. Optimal st aining conditions were evaluated for the immunocytochemical detection of vimentin in cryosections of rat liver, for DNA in situ hybridizatio n (alphoid type probes and 40-KB cosmid probes), and for RNA in situ h ybridization (detection of 28S ribosomal RNA, human elongation factor mRNA, and luciferase mRNA). Using a time-resolved fluorescence microsc ope, intense europium fluorescence was obtained in all these applicati ons when the tyramide amplification system was applied. The signals we re strong enough to be observed by eye using the microscope in the tim e-delayed mode. The routine application of this technique for localiza tion and quantization of antigens or nucleic acid sequences in tissue exhibiting strong autofluorescence is discussed.