Spectral confocal microscopy fv1000

Cardiomyocytes from either wild‐type or knock‐in Cx43 mice were isolated, plated in cover slips and incubated with the corresponding primary antibodies as previously described. Preparations were then tested using the DuoLink kit secondary antibodies conjugated with the corresponding proximity ligation assay (PLA) probes (DUO92101; Sigma‐Aldrich) for 1 hr 37°C. For ligation and amplification reactions, DuoLink in situ detection kit recommendations were followed. Cell images were obtained with spectral microscope (Olympus Spectral Confocal Microscopy FV1000) as stated above. Positive cross‐reactivity was quantified in background‐subtracted images using BlobFinder software (developed by: Carolina Wählby and Amin Allalou at CBA, Uppsala University) 25.

Isolated cardiomyocytes were fixed and permeabilized with ice‐cold acetone during 8 min. at RT. After washing with PBS + 0.025% Triton X‐100, samples were blocked with 1% BSA in PBS for 45 min. at RT and immunodetected using rabbit anti‐Cx43 antibody (C6219; Sigma‐Aldrich), mouse anti‐AIF antibody (MA5‐15880; Pierce‐Thermo Scientific), rabbit anti‐ETFB antibody (17925‐1‐AP; ProteinTech), mouse anti‐SDHA (MS204; MitoSciences) and rabbit anti‐VDAC antibody (ab15895; AbCam, Cambridge, UK) as primary antibodies and AlexaFluor 488‐conjugated antimouse (A‐11001; Molecular Probes, Thermo Scientific, Rockford, IL, USA) and AlexaFluor 594‐conjugated anti‐rabbit (A‐11012; Molecular Probes) as secondary antibodies. Finally, cardiomyocytes were washed twice with PBS and nuclei were stained with Hoeschst 33342 (10 μg/ml). Confocal fluorescent images were captured with a spectral microscope (Olympus Spectral Confocal Microscopy FV1000, Olympus, Center Valley, PA, USA). Images were captured taken into account that the pixel size was set to 1/2 of the optical resolution, and quantitative data were obtained with ImageJ software analysis (JACoP plug‐in) 24.

Spontaneous SR Ca²⁺ sparks were analyzed in intact fluo-4 loaded cardiomyocytes incubated in control HEPES buffer using a spectral confocal microscope (Ex:488 nm, Olympus Spectral Confocal Microscopy FV1000, Olympus). Spark frequency (sparks × (100 μm⁻¹) × (s⁻¹)), amplitude (ΔF/F₀), rate (ΔF/F₀ × (s⁻¹)) and morphology (full width at half-maximal amplitude, μm) were quantified using Spark Master plugin of ImageJ software (NIH, Bethesda, MD, USA).