de Haas R R, Verwoerd N P, van der Corput M P, van Gijlswijk R P, Siitari H, Tanke H J
Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.
J Histochem Cytochem. 1996 Oct;44(10):1091-9. doi: 10.1177/44.10.8813073.
The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quantum yield. To increase the intensity of the delayed fluorescence, we have used a recently introduced peroxidase-mediated amplification system. This system results in a large accumulation of biotin-tyramide, which is detected using streptavidin-europium chelate as label. Optimal staining conditions were evaluated for the immunocytochemical detection of vimentin in cryosections of rat liver, for DNA in situ hybridization (alphoid type probes and 40-KB cosmid probes), and for RNA in situ hybridization (detection of 28S ribosomal RNA, human elongation factor mRNA, and luciferase mRNA). Using a time-resolved fluorescence microscope, intense europium fluorescence was obtained in all these applications when the tyramide amplification system was applied. The signals were strong enough to be observed by eye using the microscope in the time-delayed mode. The routine application of this technique for localization and quantization of antigens or nucleic acid sequences in tissue exhibiting strong autofluorescence is discussed.
铕螯合物作为荧光原位杂交（FISH）和免疫细胞化学中延迟荧光标记物的应用，因其相对较低的量子产率而受到阻碍。为了增强延迟荧光的强度，我们使用了最近引入的过氧化物酶介导的扩增系统。该系统会导致生物素酪胺大量积累，使用链霉亲和素 - 铕螯合物作为标记物来检测。我们评估了大鼠肝脏冰冻切片中波形蛋白免疫细胞化学检测、DNA原位杂交（α卫星型探针和40 - KB黏粒探针）以及RNA原位杂交（检测28S核糖体RNA、人延伸因子mRNA和荧光素酶mRNA）的最佳染色条件。使用时间分辨荧光显微镜，当应用酪胺扩增系统时，在所有这些应用中都获得了强烈的铕荧光。信号强度足以在延时模式下通过显微镜肉眼观察到。本文讨论了该技术在具有强自发荧光的组织中抗原或核酸序列定位和定量的常规应用。
