Bertolesi G E, Trigoso C I, Espada J, Stockert J C
Department of Biology, Faculty of Exact and Natural Sciences, University of Buenos Aires, Argentina.
J Histochem Cytochem. 1995 May;43(5):537-43. doi: 10.1177/43.5.7730592.
We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma showed intense orange fluorescence and no fading under blue or blue-violet exciting light. Staining at low pH (< 2) or pre-treatment with Al3+ ions strongly diminished the fluorescence of LN, whereas that of MCG was less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharides stained with Rubipy showed high emission, which was reduced in DNA and pectic acid staining at low pH. Studies with chemically modified heparins suggested that O-sulfates were more important than N-sulfates in Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubipy staining, which could be of great value for histopathological studies. This procedure allowed visualization of the mast cells by fluorescence microscopy, and nuclei and tissue morphology were easily visualized under brightfield illumination.
我们描述了将三（2,2'-联吡啶）钌（II）（Rubipy）用作阳离子荧光染料用于细胞化学和组织化学研究。用Rubipy染色后，来自小鼠腹腔和人乳腺癌的肥大细胞颗粒（MCG）和淋巴细胞核（LN）在蓝色或蓝紫色激发光下呈现强烈的橙色荧光且不褪色。在低pH值（<2）下染色或用Al3+离子预处理会强烈降低LN的荧光，而MCG的荧光受影响较小。Ca2+和Ba2+离子仅降低MCG的荧光。用Rubipy染色的DNA、果胶酸、肝素和其他硫酸化多糖的印迹显示出发射强度较高，在低pH值下DNA和果胶酸染色的发射强度降低。对化学修饰肝素的研究表明，在Rubipy与肝素的相互作用中，O-硫酸盐比N-硫酸盐更重要。这些结果与Rubipy和肝素之间的离子结合模式一致。在Rubipy染色前用Mayer苏木精发现了一种非常适合检测肥大细胞的方法，这对组织病理学研究可能具有重要价值。该程序允许通过荧光显微镜观察肥大细胞，并且在明场照明下很容易观察到细胞核和组织形态。
