EdU Labeling for Cell Proliferation

For EdU labeling, dams were injected with EdU (15 mg/kg, Ribobio) that was diluted in PBS 2 h prior to embryo harvesting, collected in ice-cold PBS, and fixed overnight in 4% PFA at 4°C overnight. The tissues were then incubated with an ascending series of sucrose concentrations from 10% to 25% and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek United States Inc., Torrance, CA, United States). Cryosections (10 μm) were prepared, and immunostaining was performed as previously described (Fan et al., 2020 (link)). The cell proliferation rate was measured using the Cell-Light EdU Apollo488 in vitro Kit (Ribobio, C10310-3) and the immunofluorescence staining using the antibody recognizing phospho-Histone 3 (antibody from Millipore, 2465253). Cell apoptosis was assessed by immunofluorescence staining using the antibody recognizing cleaved-Caspase 3 (Cell Signaling Technologies, 9661). α-Actinin (Sigma, A7811) and PECAM1 (BD Biosciences, 550274) were used to stain cardiac and endothelial cells, respectively. DAPI was used to counterstain nuclei. The slides were imaged and subjected to an independent blinded analysis, using the Olympus IX73 confocal microscope and ImageJ software.