In Vivo and In Situ Replication Labeling Methods for Super-resolution Structured Illumination Microscopy of Chromosome Territories and Chromatin Domains
- PMID: 27283306
- DOI: 10.1007/978-1-4939-3631-1_10
In Vivo and In Situ Replication Labeling Methods for Super-resolution Structured Illumination Microscopy of Chromosome Territories and Chromatin Domains
Abstract
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.
Keywords: Chromatin; Chromosome territories; F-ara-EdU; Replication domains; Replication labeling; Structured illumination microscopy; Super-resolution imaging.
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