Cells were fixed with 4% ice-cold paraformaldehyde (PFA) (Sigma) for 15 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS (Gibco) for 10 min, followed by 30 min of blocking in 1% BSA (Roche). The cells were then stained overnight at 4 °C with the following primary antibodies: sarcomeric α-actinin (1:400; Sigma), LRP6 (1:100; Abcam), LRP5 (1:100; Abcam), Cx43 (1:200; Sigma), PDI (1:100; Novus), GM130 (1:100; CST), α-tubulin (1:400; Sigma), Cx45 (1:200; Abcam), Cx40 (1:200; Abcam) and β-catenin (1:100; Sigma). Next, the cells were washed with PBS and incubated for 1 h with the respective secondary antibodies conjugated to Alexa Fluor 488/555/633 (1:300; Invitrogen), after which the cells were stained with ToTo-3 (Invitrogen) for an additional 20 min if needed.
Sections of mouse and rat hearts were deparaffinized and rehydrated, and then boiled in sodium citrate solution for 20 min for antigen retrieval. Slides were processed for immunofluorescence visualization according to the procedure used for cultured cardiomyocytes. Images were captured using a Leica SP5 laser confocal microscope with a × 63 oil lens. Co-localization was analysed using Image-Pro Plus software (MediaCybernetics).
Sections of mouse and rat hearts were deparaffinized and rehydrated, and then boiled in sodium citrate solution for 20 min for antigen retrieval. Slides were processed for immunofluorescence visualization according to the procedure used for cultured cardiomyocytes. Images were captured using a Leica SP5 laser confocal microscope with a × 63 oil lens. Co-localization was analysed using Image-Pro Plus software (MediaCybernetics).