Immunofluorescence Staining of Cardiomyocytes

Differentiated EBs (D8) were cultured on gelatinized 12-mm glass cover slips, fixed for 30 min at room temperature in 4% paraformaldehyde, and permeabilized for 30 min at room temperature using 0.2% Triton X-100. After permeabilization, the EBs were blocked for 10 min at room temperature with CAS-Block solution (Invitrogen), and incubated overnight at 4 °C with mouse monoclonal antibodies against sarcomeric α-actinin (Abcam), cardiac troponin T (cTnT, Abcam), or connexin 43 (Cx43, Abcam). Additionally, goat anti-mouse IgG (Abcam) was used as the primary antibody to label EBs. Confocal images were obtained using a Carl Zeiss LSM 700 laser-scanning microscope (Zeiss, Oberkochen, Germany) [19 (link)].