Immunostaining for DNA modifications: computational analysis of confocal images

Ramsawhook, Ashley and Lewis, Lara C. and Eleftheriou, Maria and Abakir, Abdulkadir and Durczak, Paulina M. and Markus, Robert and Rajini, Seema and Hannan, Nicholas R.F. and Coyle, Beth and Ruzov, Alexey (2017) Immunostaining for DNA modifications: computational analysis of confocal images. Journal of Visualized Experiments . ISSN 1940-087X (In Press)

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Abstract

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Item Type: Article
Keywords: Epigenetics, DNA, oxi-mCs, N6-methyladenine, Immunohistochemistry, Zen, Confocal Microscopy, Signal Quantitation, Spatial Distribution
Schools/Departments: University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Medicine > Division of Cancer and Stem Cells
Depositing User: Eprints, Support
Date Deposited: 12 May 2017 14:38
Last Modified: 12 May 2017 16:19
URI: http://eprints.nottingham.ac.uk/id/eprint/42816

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