Immunohistochemical detection of global epigenetic DNA modifications using antibodies to 5-methylcytosine and 5-hydroxymethylcytosine. Impact of antigen retrieval protocols.

Abstract The aim of this study was to compare three different pretreatment protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. Several types of human biological samples were analyzed, including formalin fixed, paraffin embedded (FFPE) normal squamous epithelium, ethanol fixed cultured cells and metaphase chromosomes. The antigen retrieval methods included the low pH citrate and high pH Tris/EDTA protocols, as well as a method including a pepsin pretreatment step combined with an HCl DNA denaturation step. We saw a gradual increase in the detection levels of 5-mC and 5-hmC when going from citrate via Tris/EDTA to pepsin/HCl retrieval. While the citrate retrieval protocol was the least efficient for the immunochemical detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled the visualization of intra- and internuclear differences in FFPE tissue and cell culture samples. By simultaneous fluorescent detection, we assessed the differences in distribution patterns for both 5-mC and 5-hmC. In addition, (hydroxy)methylation levels in FFPE material were quantified using confocal and non-confocal microscopic imaging. As a result, we observed a significant heterogeneity, as well as differences between the levels for 5-mC and 5-hmC, within and between nuclei in the different compartments of normal squamous epithelium.