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Antibody based detection of global nuclear DNA methylation in cells, tissue sections and mammalian embryos

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

Original languageEnglish
Title of host publicationDNA Methylation Protocols
PublisherSpringer
Publication statusE-pub ahead of print - 10 Dec 2017

Publication series

NameMethods in Molecular Biology

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 immunolabelling 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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