Electron microscopy

Hearts were transcardially perfused and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer. After three washes with 0.1 M sodium cacodylate buffer, hearts were post-fixed in 1% osmium tetroxide and 0.8% K3 [Fe(CN₆)]/0.1 M sodium cacodylate buffer (1 hour, RT). After three rinses with water, specimens were dehydrated with increasing concentrations of ethanol, infiltrated with Embed-812 resin, and polymerized in a 60°C oven overnight. Blocks were sectioned with a diamond knife (Diatome) on a Leica Ultracut UC7 ultramicrotome (Leica Microsystems) and collected onto copper grids, post-stained with 2% uranyl acetate in water and lead citrate. Images were acquired on a Tecnai G2 spirit transmission electron microscope (FEI) equipped with a LaB6 source (120 kV).