Abstract
Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry.
Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin.
Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.
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Acknowledgments
We thank Tatiana Chebotareva, Cristina Aguilar, and other members of the S.P lab for their help in developing some of the protocols presented, and we thank Richard Meehan for helpful suggestions. All present and past members from N. B’s lab are acknowledged for their work and contributions, especially Claire Boulesteix. We wish to acknowledge the Niveleau lab for their development of the original 5mC antibody and some of the protocols outlined in this chapter. N.A.A.H. is a recipient of a MARA (Malaysia) scholarship; work in S.P.’s lab is supported by the BBSRC and the BHF. Work in N.B.’s lab is supported by the REVIVE Labex (Investissement d'Avenir, ANR-10-LABX-73).
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Beaujean, N., Salvaing, J., Hadi, N.A.A., Pennings, S. (2018). Antibody-Based Detection of Global Nuclear DNA Methylation in Cells, Tissue Sections, and Mammalian Embryos. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_4
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DOI: https://doi.org/10.1007/978-1-4939-7481-8_4
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