Characterization of CEOCs and CDCs by immunofluorescence analysis

Immunofluorescence analysis of CEOCs and CDCs were carried out according to the established protocol of our lab [26, 27]. Cardiac explants and CDCs were cultured directly on fibronectin coated glass cover slips for immunofluorescence analysis. CEOCS obtained from explants were analyzed for c-kit marker, specific to CPCs and CDCs were analyzed for surface markers, c-kit and CD 105 (specific to CDCs). Cells were fixed with 4% paraformaldehyde for 15 minutes and blocked in blocking buffer (1% BSA in PBS) for 30 minutes at room temperature. Cells were then incubated with primary antibodies, c-kit and CD 105 (1:200 dilution in blocking buffer) overnight at 4°C. Following three PBS washes, cells were incubated with respective fluorescence conjugated secondary antibodies (1:1000) for 1 hour at room temperature. DAPI was used to counterstain nuclei and the cells were visualized using confocal microscope (Carl Zeiss, Zen 2010) with appropriate filters.