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

Methods Mol Biol. 2016:1431:127-40. doi: 10.1007/978-1-4939-3631-1_10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Nucleus / genetics
  • Chromatin / metabolism
  • Chromatin / ultrastructure*
  • Chromosome Segregation
  • Chromosomes, Mammalian / genetics
  • Chromosomes, Mammalian / ultrastructure*
  • DNA Replication
  • Imaging, Three-Dimensional / methods*
  • Mice
  • Microscopy, Fluorescence
  • Replication Origin

Substances

  • Chromatin