DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes

Sonia Heras; Katrien Forier; Koen Rombouts; Kevin Braeckmans; Ann Van Soom

doi:10.1016/j.ab.2014.03.002

DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes

Anal Biochem. 2014 Jun 1:454:14-6. doi: 10.1016/j.ab.2014.03.002. Epub 2014 Mar 11.

Authors

Sonia Heras¹, Katrien Forier², Koen Rombouts², Kevin Braeckmans², Ann Van Soom²

Affiliations

¹ Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. Electronic address: sonia.herasgarcia@ugent.be.
² Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.

PMID: 24631518
DOI: 10.1016/j.ab.2014.03.002

Abstract

Immunostaining is the preferred technique to assess differences in methylation and hydroxymethylation status of both pronuclei in single zygotes. DNA counterstaining is needed to delimitate the pronuclear area for quantification purposes. For a correct epitope retrieval of 5-methylcytosine and 5-hydroxymethylcytosine in bovine zygotes, 1h of denaturation with 4N HCl is needed. However, DNA stains are sensitive to denaturation. Therefore, four DNA stains were tested after 1h of denaturation with 4N HCl in this study. After this treatment, DAPI (4',6-diamidino-2-phenylindole) and Hoechst failed to bind DNA, but both propidium iodide and ethidium homodimer-2 successfully bound it and both pronuclei were stained.

Keywords: Bovine zygotes; DNA counterstaining; Epigenetics; Hydroxymethylation; Immunofluorescence; Methylation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

5-Methylcytosine / metabolism*
Animals
Cattle
Cytosine / analogs & derivatives*
Cytosine / metabolism
DNA Methylation
Staining and Labeling / methods*
Zygote / metabolism*

Substances

5-hydroxymethylcytosine
5-Methylcytosine
Cytosine