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In vivo immunogold labeling confirms large-scale chromatin folding motifs

Nature Methods volume 5, pages 311–313 (2008)Cite this article

Abstract

The difficulty in localizing specific cellular proteins by immuno-electron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP–proliferating cell nuclear antigen (PCNA).

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Figure 1: Demonstration of in vivo immunogold labeling using A03 cells.
Figure 2: In vivo conformation of large-scale chromatin fibers.
Figure 3: In vivo labeling of DNA replication foci reveals GFP-PCNA within condensed, large-scale chromatin domains.

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Acknowledgements

This work was supported by the US National Institute of General Medical Sciences. (grant GM42516 to A.S.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

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Author notes

  1. Margot Lakonishok

    Present address: Present address: Department of Molecular and Cell Biology, Northwestern Medical School, 303 E. Chicago Ave., Ward 11-080, Chicago, Illinois 60611, USA.,

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, 601 S. Goodwin Ave., Urbana, 61801, Illinois, USA

    Igor Kireev, Margot Lakonishok & Andrew S Belmont

  2. Department of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Building A, Leninskie Gory, GSP-1, Moscow, 119991, Russia

    Igor Kireev

  3. Nanoprobes, Inc., 95 Horse Block Road, Unit 1, Yaphank, 11980, New York, USA

    Wenqiu Liu, Vishwas N Joshi & Rick Powell

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  1. Igor Kireev
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  2. Margot Lakonishok
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Contributions

I.K. performed in vivo labeling and electron microscopy analysis; M.L. developed the monoclonal antibodies, purified these antibodies and prepared Fab′ fragments, and carried out initial in vivo labeling feasibility experiments; R.P., W.L. and V.N.J. optimized and conducted the Nanogold antibody labeling, in conjunction with M.L.; A.S.B. supervised the overall project.

Corresponding author

Correspondence to Andrew S Belmont.

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Competing interests

W.L., V.N.J. and R.P. are employees of Nanoprobes, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Methods (PDF 1764 kb)

Supplementary Movie 1

Tilted projections showing gene amplified region from PDC cell labeled in vivo with anti-GFP Nanogold antibodies. Large-scale chromatin fibers of 120-170 nm diameter can be visualized corresponding to the GFP-lac repressor staining of the lac operator containing, amplified transgene array. Projection step size 5 degrees. Scale bar, 500 nm. (MOV 1820 kb)

Supplementary Movie 2

Rocking projections of a mid-S phase, DNA replication focus, as identified by GFP-PCNA labeled in vivo with anti-GFP Nanogold antibodies. Note the appearance of a uniform distribution of label through the replication focus. Projection step size 5 degrees. Cell was permeabilized prior to fixation to reveal surrounding chromatin (darkly stained material). (MOV 1281 kb)

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Kireev, I., Lakonishok, M., Liu, W. et al. In vivo immunogold labeling confirms large-scale chromatin folding motifs. Nat Methods 5, 311–313 (2008). https://doi.org/10.1038/nmeth.1196

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