Detection of Global DNA Methylation in the Hearts of Zebrafish Larvae

DNA methylation is one of the most extensively studied epigenetic modifications and plays an essential role in cardiac development. The zebrafish is a popular model for studying heart development due to its genetic similarity to mammals and transparent embryos. However, its small size presents challenges in obtaining sufficient DNA for methylation analysis. In this study, zebrafish embryos were exposed to 5-Azacytidine (5-AzaC) at concentrations of 0 µM, 25 µM, and 50 µM from 2-72 hours post-fertilization (2-72 hpf). Hearts were isolated from whole embryos, fixed, and permeabilized. The samples were then treated with proteinase K and HCl to facilitate access to DNA, followed by probing with an anti-5-methylcytosine (anti-5 mC) antibody. After incubation with a secondary antibody, the hearts were co-stained with DAPI. Fluorescence images were captured using a microscope and analyzed with ImageJ. Results demonstrated that 5-AzaC treatment significantly reduced global DNA methylation levels in zebrafish embryonic hearts. In conclusion, we present a sensitive immunofluorescence method to detect global DNA methylation levels within a single zebrafish larvae heart. This technique can be used to evaluate DNA methylation changes induced by environmental pollutants and to explore their impacts on heart development.