Immunofluorescence Staining of Cardiomyocytes

The cells were fixed in 4% paraformaldehyde for 10 minutes and then permeabilized in 0.4% Triton X-100 for 6 minutes. The fixed cells were blocked with 5% normal goat serum for 1 hour at room temperature. Samples were incubated overnight at 4°C with following primary antibodies: mouse polyclonal anti-α-ACTININ (1:400, Sigma), rabbit polyclonal anti-connexin-43 (0.5μg/ml, Boster Biological). The samples were rinsed with PBS and stained for 1 hour with the following secondary antibodies: goat anti-mouse IgG Alexa Fluor488, goat anti-rabbit IgG Alexa Fluor594 (all from Jackson Immuno Research Laboratories, West Grove, PA, 1:1,000). All samples were counterstained with Hoechst 3342 (1μg/ml, Sigma-Aldrich) for 3 min. A Zeiss Observer microscope was used for slide observation and image capture. Sarcomere lengths were measured using Image J software. We analyzed the hESC-CM sarcomere length (measured by the distance between Z-disks) and the cell circularity index (Circularity = 4π·Area / Perimeter²) using previously published methods [43 (link), 70 (link), 71 (link)].