In Vivo and In Situ Replication Labeling Methods for Super-resolution Structured Illumination Microscopy of Chromosome Territories and Chromatin Domains.

Autor:	Miron E; Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK., Innocent C; Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK., Heyde S; Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK., Schermelleh L; Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. lothar.schermelleh@bioch.ox.ac.uk.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2016; Vol. 1431, pp. 127-40.
DOI:	10.1007/978-1-4939-3631-1_10
Abstrakt:	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.
Databáze:	MEDLINE
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