Simplified and Optimized Kinetochore Detection: Cytogenetic Marker for Late-G sub2 Cells

摘要

Cytogenetic detection of kinetochore proteins using the CREST antibody coupedwith 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-XiOO, 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