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

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Chromosome Architecture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2476))

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.

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Acknowledgments

This work was supported by the Wellcome Trust Strategic Award 091911, funding advanced microscopy at Micron Oxford, and the John Fell Oxford University Press (OUP) Research Fund 143/064. Fena Ochs was supported by a DFF International Postdoc Fellowship (0164-00011B).

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Authors and Affiliations

  1. Department of Biochemistry, University of Oxford, Oxford, UK

    Ezequiel Miron, Joseph Windo, Fena Ochs & Lothar Schermelleh

Authors
  1. Ezequiel Miron
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  2. Joseph Windo
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  3. Fena Ochs
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  4. Lothar Schermelleh
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Corresponding author

Correspondence to Lothar Schermelleh .

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Editors and Affiliations

  1. Departments of Physics and Biology, University of York, York, UK

    Mark C. Leake

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Miron, E., Windo, J., Ochs, F., Schermelleh, L. (2022). Replication Labeling Methods for Super-Resolution Imaging of Chromosome Territories and Chromatin Domains. In: Leake, M.C. (eds) Chromosome Architecture. Methods in Molecular Biology, vol 2476. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2221-6_9

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  • DOI: https://doi.org/10.1007/978-1-0716-2221-6_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2220-9

  • Online ISBN: 978-1-0716-2221-6

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