Mouse monoclonal igg

For confocal fluorescence imaging of the structural proteins for myocyte morphology, the hearts were fixed using Langendorff perfusion with 2% paraformaldehyde for 30 min (n = 29 hearts). We used Alexa Fluor 633 phalloidin (dilution 1:400, Life Technologies) for actin histochemistry. For sarcomeric α-actinin immunohistochemistry, incubation with a primary antibody (dilution 1:500, mouse monoclonal IgG, Sigma‒Aldrich, USA) for 48 h at 4 °C was followed by incubation with a secondary antibody for 48 h (dilution 1:500, Alexa Fluor 488, anti-mouse, Thermo Fisher Scientific, USA) prior to visualisation at room temperature. Confocal fluorescence images of both phalloidin and α-actinin were obtained using samples from the subepicardial surface of the heart using an FV-1000 confocal microscope (Olympus, Japan). The excitation wavelengths for phalloidin and sarcomeric α-actinin were 633 nm and 488 nm, respectively, and the emission wavelengths were > 650 nm and between 500 and 600 nm, respectively. To confirm membrane permeabilisation by saponin, we perfused in advance the membrane dye di-4-ANEPPS at 5 μM (Wako Pure Chemicals, Japan) and the membrane-impermeable DNA dye propidium iodide (PI) at 7.5 μM (Dojindo, Japan) for 5 min and subsequent washout for another 5 min for confocal imaging (n = 3 hearts).