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.
CITATION STYLE
Miron, E., Windo, J., Ochs, F., & Schermelleh, L. (2022). Replication Labeling Methods for Super-Resolution Imaging of Chromosome Territories and Chromatin Domains. In Methods in Molecular Biology (Vol. 2476, pp. 111–128). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2221-6_9
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