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Recent advances in super-resolution microscopy enable the study of subchromosomal 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) for the in situ detection of segregated chromosome territories with minimized cytotoxic side effects.

Original publication

DOI

10.1007/978-1-4939-3631-1_10

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2016

Volume

1431

Pages

127 - 140

Keywords

Chromatin, Chromosome territories, F-ara-EdU, Replication domains, Replication labeling, Structured illumination microscopy, Super-resolution imaging