Journal of Visualized Experiments, volume 2022, issue 183
Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography
Publication type: Journal Article
Publication date: 2022-05-20
Journal:
Journal of Visualized Experiments
Quartile SCImago
Q2
Quartile WOS
Q3
Impact factor: 1.2
ISSN: 1940087X
DOI:
10.3791/62803
General Biochemistry, Genetics and Molecular Biology
General Chemical Engineering
General Immunology and Microbiology
General Neuroscience
Abstract
Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ, by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.
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Sosnovskaya S. et al. Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography // Journal of Visualized Experiments. 2022. Vol. 2022. No. 183.
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Sosnovskaya S., Zakirov A. N., Ryumina E. D., Kharybina E., Golyshev S. A., Strelkova O. S., Zhironkina O. A., Moiseenko A., Orekhov A., Kireev I. I. Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography // Journal of Visualized Experiments. 2022. Vol. 2022. No. 183.
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TY - JOUR
DO - 10.3791/62803
UR - https://doi.org/10.3791%2F62803
TI - Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography
T2 - Journal of Visualized Experiments
AU - Sosnovskaya, Sophie
AU - Zakirov, Amir N
AU - Ryumina, Ekaterina D
AU - Kharybina, Ekaterina
AU - Golyshev, Sergei A
AU - Strelkova, Olga S
AU - Zhironkina, Oxana A
AU - Moiseenko, Andrei
AU - Orekhov, Anton
AU - Kireev, Igor I
PY - 2022
DA - 2022/05/20 00:00:00
PB - MYJoVE Corporation
IS - 183
VL - 2022
SN - 1940-087X
ER -
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@article{2022_Sosnovskaya,
author = {Sophie Sosnovskaya and Amir N Zakirov and Ekaterina D Ryumina and Ekaterina Kharybina and Sergei A Golyshev and Olga S Strelkova and Oxana A Zhironkina and Andrei Moiseenko and Anton Orekhov and Igor I Kireev},
title = {Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography},
journal = {Journal of Visualized Experiments},
year = {2022},
volume = {2022},
publisher = {MYJoVE Corporation},
month = {may},
url = {https://doi.org/10.3791%2F62803},
number = {183},
doi = {10.3791/62803}
}