Immunofluorescence Staining of iPSC-CMs

Ctrl- and RYR2^–/–-iPSC-CMs grown on glass coverslips were fixed with 4% paraformaldehyde (PFA; Carl Roth), blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich), and permeabilized with 0.1% Triton X-100 (Carl Roth). Immunofluorescence staining was performed overnight using the following primary antibodies: anti-α-actinin (1:500; mouse monoclonal, IgG1, Sigma-Aldrich), anti-Ki67 (1:400; rabbit polyoclonal, abcam), anti-IP3R (1:100; rabbit polyclonal, Merck Millipore, used for all three subtypes of IP3R), and anti-RYR2 (1:500; rabbit polyclonal, HPA020028; Sigma-Aldrich, used for the full length of RYR2). Afterward, cells were washed three times with PBS and incubated with the corresponding secondary antibodies (1:1000; anti-rabbit Alexa fluor 546, Invitrogen; anti-rabbit Alexa fluor 488, Invitrogen; anti-mouse Alexa fluor 546, Invitrogen; anti-mouse Alexa fluor 488, Invitrogen) for 1 h at RT. Nuclei were co-stained with 4’,6-diamidino-2-phenylindole (DAPI; 0.4 μg/ml; Sigma-Aldrich). Documentation was performed using fluorescence microscopy (Carl Zeiss).