Mouse anti n cadherin antibody

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).

Cells were fixed in 4% paraformaldehyde and blocked in 5% skim milk/phosphate buffer saline with 0.1% Tween-20 (PBST). After washing in PBST, cell samples were incubated with rabbit anti-FAK(p397) antibody (Abcam, Cambridge, UK) or mouse anti-N-cadherin antibody (BD Biosciences, San Jose, CA, USA) in 5% skim milk/PBST, washed with PBST twice, and incubated with Akexa488-conjugated anti-rabbit IgG antibody or Alexa546-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). Cell specimens were also stained with rhodamine phalloidin (Cytoskelton, Inc., Denver, CO, USA). Mounted cell specimens were analyzed with a confocal microscope (ZSM710; Carl Zeiss, Jena, Germany).