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Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry

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DNA Modifications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2198))

Abstract

Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.

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Acknowledgments

We thank Andrew Jackson (School of Life Sciences, Nottingham University) for provision of mouse embryonic stem cells as well as the members of School of Life Sciences Imaging (SLIM) facility and Antony Alioui for technical assistance. A.R.’s lab is supported by Biotechnology and Biological Sciences Research Council [grant number BB/N005759/1] to A.R. A.A. is supported by Medical Research Council IMPACT DTP PhD Studentship [grant number MR/N013913/1] to A.A.

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Authors and Affiliations

  1. Division of Cancer and Stem Cells, School of Medicine, Biodiscovery Institute, University of Nottingham, Nottingham, UK

    Abdulkadir Abakir & Alexey Ruzov

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  1. Abdulkadir Abakir
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  2. Alexey Ruzov
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Correspondence to Abdulkadir Abakir .

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  1. Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK

    Alexey Ruzov

  2. School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK

    Martin Gering

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Abakir, A., Ruzov, A. (2021). Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry. In: Ruzov, A., Gering, M. (eds) DNA Modifications. Methods in Molecular Biology, vol 2198. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0876-0_14

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  • DOI: https://doi.org/10.1007/978-1-0716-0876-0_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0875-3

  • Online ISBN: 978-1-0716-0876-0

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