Elyra s 1 lsm 880

Cryosectioned heart slices, as prepared above, were blocked and permeabilized in Dako protein block (Agilent, Catalogue No.: X0909) containing 0.1% (w/v) saponin (Sigma, Catalogue No.: 47036) at room temperature for 90 min. Slices were incubated with primary antibodies (see below) diluted in the same blocking solution at 4 °C for overnight. After washing with PBS, slices were incubated with secondary antibodies (see below) at room temperature for 1 h. After washing with PBS for three times, slices were mounted with ProLong gold antifade reagent containing DAPI (ThermoFisher, Catalogue No.: P36931). Primary antibodies used were polycloncal rabbit anti-Cx43 (1:200, Sigma, Catalogue No.: C6219), and monoclonal mouse anti-α-sarcomeric actinin (1:400, Sigma, Catalogue No.: A7811). Secondary antibodies used were anti-rabbit IgG (Alexa Fluor-488, 1:300) and anti-mouse IgG (Alexa Fluor-568, 1:300). Fluorescent images of the myocardium of left ventricular free wall of sham hearts and border zone of MI-hearts were taken with a ZEISS confocal microscope equipped with the Airyscan technique for high-resolution imaging (Zeiss Elyra S.1 LSM 880).

iPSC‐CMs cultured on 8‐chamber culture slides (ThermoFisher, 154461PK) were fixed with 100% methanol at −20°C for 10 min. Cells were blocked and permeabilized in Dako protein block (Agilent, X0909) containing 0.1% (w/v) saponin (Sigma‐Aldrich, 47036) at room temperature for 90 min. Cells were then incubated with primary antibodies (see below) diluted in the same blocking solution at 4°C for overnight. Then cells were washed with phosphate‐buffered saline (PBS) and incubated with secondary antibodies (see below) at room temperature for 1 h. Cells were washed with PBS and mounted with ProLong gold antifade reagent containing DAPI (ThermoFisher, P36931). Primary antibodies used were monoclonal rabbit anti‐Na_v1.5 antibody targeting the C‐terminus (1:100, Cell Signaling, 14421), monoclonal mouse anti‐α‐sarcomeric actinin antibody (1:400, Sigma, A7811), rabbit anti‐β‐catenin antibody (1:100, Cell Signaling, 8480), and mouse anti‐N‐Cadherin antibody (BD Bioscience, 610920). Secondary antibodies used were goat anti‐mouse IgG Alexa Fluor‐568 (1:300, ThermoFisher, A‐11004) and anti‐rabbit IgG Alexa Fluor‐488 (1:300, ThermoFisher, A‐11008). Fluorescent cellular images were taken with a ZEISS confocal microscope equipped with the Airyscan technique for high‐resolution imaging (Zeiss Elyra S.1 LSM 880).