title: DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes.
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.
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. Electronic address: sonia.herasgarcia@ugent.be.
authors.forename: Sonia
authors.initials: S
authors.lastname: Heras
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Forier
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Rombouts
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism* / Q000031:analogs & derivatives; D000818:Animals; D002417:Cattle; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation; D013194:Staining and Labeling / Q000379:methods*; D015053:Zygote / Q000378:metabolism*
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine
keywords: Bovine zygotes; DNA counterstaining; Epigenetics; Hydroxymethylation; Immunofluorescence; Methylation