%0 Journal Article
%T Replication Labeling Methods for Super-Resolution Imaging of Chromosome Territories and Chromatin Domains.
%A Miron E
%A Windo J
%A Ochs F
%A Schermelleh L
%J Methods Mol Biol
%V 2476
%N 0
%D 2022
%M 35635700
暂无%R 10.1007/978-1-0716-2221-6_9
%X Continuing progress in super-resolution microscopy enables the study of sub-chromosomal chromatin organization in single cells with unprecedented detail. Here we describe refined methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specific focus on their application to three-dimensional structured illumination microscopy (3D-SIM). We provide detailed protocols for highly efficient electroporation-based delivery or scratch loading of cell-impermeable fluorescent nucleotides for live-cell studies. Furthermore, we describe the application of (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) and 5-vinyl-2'-deoxyuridine (VdU) for the in situ detection of segregated chromosome territories and sister chromatids with minimized cytotoxic side effects.