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Immunostaining for DNA modifications: computational analysis of confocal images

Ramsawhook, Ashley; Lewis, Lara C.; Eleftheriou, Maria; Abakir, Abdulkadir; Durczak, Paulina M.; Markus, Robert; Rajini, Seema; Hannan, Nicholas R.F.; Coyle, Beth; Ruzov, Alexey


Ashley Ramsawhook

Lara C. Lewis

Maria Eleftheriou

Abdulkadir Abakir

Paulina M. Durczak

Robert Markus

Seema Rajini

Nicholas R.F. Hannan

Associate Professor

Alexey Ruzov


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.


Ramsawhook, A., Lewis, L. C., Eleftheriou, M., Abakir, A., Durczak, P. M., Markus, R., …Ruzov, A. (2017). Immunostaining for DNA modifications: computational analysis of confocal images. Journal of Visualized Experiments, Article e56318.

Journal Article Type Article
Acceptance Date May 5, 2017
Publication Date Jul 9, 2017
Deposit Date May 12, 2017
Publicly Available Date Jul 9, 2017
Journal Journal of Visualized Experiments
Electronic ISSN 1940-087X
Publisher Journal of Visualized Experiments
Peer Reviewed Peer Reviewed
Issue 127
Article Number e56318
Keywords Epigenetics, DNA, oxi-mCs, N6-methyladenine, Immunohistochemistry, Zen, Confocal Microscopy, Signal Quantitation, Spatial Distribution
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