ZFIN ID: ZDB-PUB-200822-22
Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos
Jessop, P., Gering, M.
Date: 2021
Source: Methods in molecular biology (Clifton, N.J.)   2198: 193-208 (Chapter)
Registered Authors: Gering, Martin
Keywords: 5-Carboxycytosine, 5-Hydroxymethylcytosine, 5caC, 5hmC, Embryo, Immunohistochemistry, Tyramide, Zebrafish
MeSH Terms:
  • 5-Methylcytosine/analogs & derivatives*
  • 5-Methylcytosine/chemistry
  • 5-Methylcytosine/metabolism
  • Animals
  • Antibodies/metabolism
  • Cell Nucleus/metabolism
  • Cytosine/analogs & derivatives
  • DNA/genetics
  • DNA/immunology*
  • DNA Methylation
  • Dioxygenases
  • Embryo, Nonmammalian
  • Immunohistochemistry/methods*
  • Zebrafish
PubMed: 32822033 Full text @ Meth. Mol. Biol.
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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