W11261

EdU detection was performed using a commercially available kit (Invitrogen), according to the manufacturer's instructions. For immunostaining of Ph3, the 3 μm-thick sections were blocked with 10% normal goat serum for 60 min and then treated with antibody against Ph3 (CST, 3475S) and TPM (Sigma, T9283) overnight at 4°C. The sections were washed with phosphate buffered saline (PBS) thrice and incubated with secondary antibody (1:200; Invitrogen) for 1 h in the dark at 37°C. Nuclei were labeled with DAPI (Sigma). Images of stained tissues were captured by Olympas confocal laser scanning microscope (FluoView 1000, Japan). Proliferation indice were calculated by dividing the numbers of CM nuclei by the numbers of Ph3⁺ or EdU⁺ CMs using the Image J software.
The cardiac cell size was examined using WGA staining (Invitrogen, W11261). Briefly, heart sections were washed with PBS three times and incubated using Alexa Fluor 488 conjugated WGA according to the manufacturer's instructions. Masson’s trichrome staining was performed to assess interstitial fibrosis. Briefly, hearts from each group were embedded in paraffin after conventional processing (alcohol dehydration) and further processed for histology.

O.C.T Compound (optimal cutting temperature compound, Sakura) embedded frozen sections of the heart samples were used for WGA staining. The 10-μm heart cross-sections were reheated at room temperature for 20 min. Then heart cross-sections were gently washed with PBS 3 times for 5 min each, fixed with 4% paraformaldehyde for 15 min. The tissue sections were incubated with WGA working solution (Invitrogen #W11261, 1:100) for 2 h at room temperature and were stained with DAPI mounting media for 20 min in dark. Then the tissue sections were washed with PBS 3 times and sealed with 50% glycerin. Images were captured by fluorescent microscope (Leica, German), the area of cardiomyocytes was measured by Image J (NIH, USA), and a minimum of 400 cardiomyocytes per mouse was analyzed.