Abend M, Blakely W F, van Beuningen D
Federal Armed Forces Medical Academy, Institute of Radiobiology, Munich, Germany.
Mutat Res. 1995 Feb;334(1):39-47. doi: 10.1016/0165-1161(95)90029-2.
Cytogenetic detection of kinetochore proteins using the CREST antibody coupled with secondary antibodies labeled with different fluorescent probes has been optimized for several in vitro mammalian cell lines. This study investigated selected parameters including the influence of common fixatives (methanol, ethanol, methanol:acetic acid (3:1)), detergents (Triton-X100, Tween), fluorescent probes (CY3, BODIPY, FITC), washing protocols, and an antifading agent (paraphenylenediamine) on the detection of kinetochore proteins in control and X-ray (240 kVp)-irradiated cells. Utilizing an optimized fixation and staining protocol, a brilliant visualization of kinetochores in interphase cells was obtained in control as well as X-ray-irradiated interhase cells. Application of this improved kinetochore staining methodology readily permits discriminating cells containing either single or paired kinetochores, the latter of which are characteristic of late-G2 phase and prophase cells.
使用与不同荧光探针标记的二抗偶联的着丝粒蛋白CREST抗体对几种体外培养的哺乳动物细胞系进行细胞遗传学检测的方法已得到优化。本研究调查了一些选定参数，包括常用固定剂（甲醇、乙醇、甲醇：乙酸（3:1））、去污剂（Triton-X100、吐温）、荧光探针（CY3、BODIPY、FITC）、洗涤方案以及抗褪色剂（对苯二胺）对对照细胞和X射线（240 kVp）照射细胞中着丝粒蛋白检测的影响。利用优化的固定和染色方案，在对照细胞以及X射线照射的间期细胞中均获得了间期细胞中着丝粒的清晰可视化。应用这种改进的着丝粒染色方法能够轻松区分含有单个或成对着丝粒的细胞，后者是G2期晚期和前期细胞的特征。
