Optishot

Primary cardiomyocytes and 10‐μm RV and LV cryosections were fixed with 4% paraformaldehyde for 10 minutes at 37°C, washed twice for 5 minutes with PBS, incubated with Alexa Fluor‐488 conjugated wheat germ agglutinin (Sigma‐Aldrich) for 10 minutes at room temperature, and then washed in PBS twice for 5 minutes and mounted in Anti‐Fade Reagent (Molecular Probes). Images were collected using a Bio‐Rad (Hercules, CA) MRC 1000 scan head mounted on an upright Nikon Optishot (Tokyo, Japan) microscope at the University of Minnesota Imaging Center. Z‐stacks were collected and converted into a Z‐projection using ImageJ (National Institutes of Health, Bethesda, MD). T‐tubule structure and regularity were quantified using the TTPower plugin on ImageJ, which uses fast Fourier transformation to quantitate the regularity of t‐tubules as previously described.16, 28