Electron Microscopy.
Hearts were dissected, and 0.5- to 1-mm3 slices were fixed by immersion for 2 h in 2.5% (wt/vol) glutaraldehyde in 0.1 M cacodylate buffer and were prepared for electron microscopy by standard methods. Briefly, cells were postfixed in osmium tetroxide [1% (wt/vol) in 0.1 M phosphate buffer], stained with uranyl acetate [2% (wt/vol) in distilled water], dehydrated through increasing concentrations of ethanol (70–100%) and acetone, and embedded in TAAB resin (TAAB Laboratories Equipment, Ltd.). Ultrathin sections (50–80 nm) were prepared using a Reichert Ultracut S microtome and were mounted on 200-mesh nickel grids. Sections were lightly counterstained with lead citrate and uranyl acetate and were examined with a Jeol transmission electron microscope (JEM-1010; JEOL).