Replication Labeling Methods for Super-Resolution Imaging of Chromosome Territories and Chromatin Domains

Ezequiel Miron, Joseph Windo, Fena Ochs, Lothar Schermelleh

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

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.

Original languageEnglish
Title of host publicationChromosome Architecture
EditorsMark C. Leake
Number of pages18
PublisherSpringer
Publication date2022
Edition2
Pages111-128
ISBN (Print)978-1-0716-2223-0, 978-1-0716-2220-9
ISBN (Electronic)978-1-0716-2221-6
DOIs
Publication statusPublished - 2022
SeriesMethods in Molecular Biology
Volume2476
ISSN1064-3745

Bibliographical note

© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • Animals
  • Cell Nucleus
  • Chromatids
  • Chromatin/genetics
  • Mammals
  • Microscopy/methods

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