In vivo and in situ replication labeling methods for super-resolution structured illumination microscopy of chromosome territories and chromatin domains

Ezequiel Miron, Cassandravictoria Innocent, Sophia Heyde, Lothar Schermelleh*

*Corresponding author for this work

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

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Number of pages14
Volume1431
PublisherHumana Press
Publication date2016
Pages127-140
DOIs
Publication statusPublished - 2016
Externally publishedYes
SeriesMethods in Molecular Biology
Volume1431
ISSN1064-3745

Keywords

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

Cite this

Miron, E., Innocent, C., Heyde, S., & Schermelleh, L. (2016). In vivo and in situ replication labeling methods for super-resolution structured illumination microscopy of chromosome territories and chromatin domains. In Methods in Molecular Biology (Vol. 1431, pp. 127-140). Humana Press. Methods in Molecular Biology, Vol.. 1431 https://doi.org/10.1007/978-1-4939-3631-1_10