Jem 1010 electron microscope

After fixation of the cardiac specimens in 2.5–4% glutaraldehyde solution (pH 7.4) for 48 h at 4 °C, these specimens were cut into small particles and washed with distilled water. After that, they were fixed in 1% osmium tetraoxide with 15 mg/mL of potassium ferrocyanide for 1–2 h at 4 °C. The tissue specimens thereafter were cut with an ultramicrotome to sections of 0.5–1 μm thickness and stained with uranyl acetate and lead. After that, these sections were examined and photomicrographs captured using a JEOL, JEM 1010 electron microscope (Jeol Ltd., Tokyo, Japan).

Cardiomyocytes seeded in p35 plates with Matrigel^® and RPMI/B27 containing insulin, 10% FBS and 10 µM Y27632 were fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 2 h at room temperature. Fixed cells were included in epoxy resin (TAAB 812 resin, TAAB laboratories, Berkshire, England) by conventional methods. For this, cells were stained for 1 h with 1% osmium tetroxid + 0.08% potassium ferricianide at 4 °C and with 2% uranyl acetate in water for another hour at 4 °C. Dehydration (EtOH: 50%, 75%, 90%, 95% and 100%) was carried out at 4 °C and embedded with the resin (epoxy resin: EtOH 1:2, 1:1, 2:1, and 100% resin) at room temperature. Finally, the resin was polymerized for 48 h at 60 °C. Ultrathin 70 nm cuts were obtained on a Leica Ultracut UCT ultramicrotome (Leica, Vienna, Austria) with a DiATOME diamond blade. Sections were collected on hexagonal drawing Cu/Pd grids, and 100 windows per square inch (200 mesh) were coated with formvar and a layer of evaporated carbon. The sections were stained with 2% uranyl acetate in water for 7 min and with Reynolds lead citrate for 3 min. About 100 images from each sample were taken with a 4 K × 4 K, F416 CMOS camera from TVIPS (Gauting, Germany) at 5000× or 8000× magnification on the JEOL JEM-1010 electron microscope (JEOL, Akishima, Tokyo, Japan) at an electron acceleration voltage of 80 kV.