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Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos

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DNA Modifications

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

Abstract

5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.

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Acknowledgments

We wish to thank Adulkadir Abakir, Tim Self, Denise McClean, and Ian Ward at the University of Nottingham for their technical assistance with the immunohistochemistry and imaging. P.J. was supported by a BBSRC-DTP PhD studentship (BB/J014508/1).

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Authors and Affiliations

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

    Peter Jessop & Martin Gering

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  1. Peter Jessop
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  2. Martin Gering
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Correspondence to Martin Gering .

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Editors and Affiliations

  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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Jessop, P., Gering, M. (2021). Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos. 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_16

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  • DOI: https://doi.org/10.1007/978-1-0716-0876-0_16

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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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