Immunohistochemical Detection of Oxidized Forms of 5-Methylcytosine in Embryonic and Adult Brain Tissue
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DNA methylation (5-methylcytosine, 5mC) is a major epigenetic modification of the eukaryotic genome associated with gene repression. Ten-eleven translocation proteins (Tet1/2/3) can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Recent studies demonstrate that 5hmC is particularly enriched in neuronal cells and imply the involvement of this mark in transcriptional regulation taking place within the mammalian brain. Although a number of biochemical and antibody-based approaches have been successfully used to study the global content and genomic distributions of 5hmC in various contexts, most of these techniques do not provide any spatial information on the levels of this mark in different cell types. Here we describe a method of sensitive immunochemical detection of 5hmC/5fC/5caC in brain tissue based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification. This technique can be instrumental in determining the relative enrichments of oxidized forms of 5mC in different brain cell types, effectively complementing other established approaches to investigate the functions of these marks in embryonic and adult brain.
Key wordsDNA methylation Tet1/2/3 proteins 5-hydroxymethylcytosine 5-carboxylcytosine 5-formylcytosine Embryonic brain Adult brain Immunohistochemistry Signal amplification Immunofluorescence Microscopy
We thank Paul De Sousa (University of Edinburgh), Rimple D’Almeida, Rebecca Trueman (University of Nottingham), the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), and the team of Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) for help and support.
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