Ultracut uct ultramicrotome

The scalp specimen was trimmed into a small piece (approximately 1 mm³) and was fixed in 2.5% glutaraldehyde (buffered to pH 7.3 with sodium cacodylate) for 4 h at 4 °C, and then was washed with 0.1 mol/L cacodylate buffer. The specimen was post-fixed in 1% osmium tetroxide in 0.1 mol/L cacodylate buffer at a pH of 7.3 for 1 h. Sequentially, the specimen was dehydrated using ethanol at concentrations of 30%, 50%, 70%, 80%, 90% and 100%, each phase for 10 min. This was followed by overnight impregnation with propylene and epoxyresin (Epon 812). Ultrathin sections (70 nm thick) were obtained using a Leica Ultracut UCT ultramicrotome (EM UC6, Leica, Wetzlar, Germany). These sections were placed onto a copper grid and stained with lead and uranyl solutions, after which they were observed and photographed using a TEM (JEM-2100, JEOL, 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.

Transmission electron microscopy (TEM) was performed as previously described [16 (link)]. Immediately after excision, LV samples were fixed in 4% formahaldehyde: 1% glutaraldehyde in cacodylate buffer, and postfixed in 1% osmium tetroxide. Tissues were then washed in PBS, dehydrated through graded alcohols followed by acetone, and then infiltrated with Durcupan ACM Fluka resin and polymerized at 60 °C for 48 h. Blocks were cut with a Leica ultracut UCT ultramicrotome (Leica, Heerbrugg, Switzerland), and Sects. (60–70 nm) were mounted onto 200-mesh grids. Sections were stained with a 2% solution of aqueous uranyl acetate for 10 min, followed by lead citrate staining for 10 min. Stained sections were viewed with a JEOL JEM-1010 transmission electron microscope (Tokyo, Japan) operating at 80 kV through 6000 × , 10,000 × , and 40,000 × objectives. Images were acquired with a GATAN Orius 200SC digital camera. Mitochondrial morphometry was analyzed using ImageJ (National Institutes of Health).

Cell membrane integrity was evaluated by transmission electron microscopy (TEM) analysis. Briefly, cells subjected to different treatments (BP, HP, HP-ES, and ES) were centrifuged and collected after washing twice with PBS buffer (0.01 M, pH 7.0). In total, 1 mL 2.5% glutaraldehyde was added to fix cells for 3 h at room temperature before being washed twice with PBS buffer (0.01 M, pH 7.0). Subsequently, the cells were washed, dehydrated, and embedded. Finally, ultrathin sections (70–80 nm) obtained by a Leica Ultracut UCT ultramicrotome (Leica) were captured using an electron microscope (JEM-1400, Japan) at room temperature.