Diamond histoknife

Transmission electron microscopy was used to investigate the ultrastructural changes in mitochondria. After 0 h, 24 h or 48 h of hypothermic preservation, the renal tissue was diced into 1 mm³ pieces and further fixed using 2.5% glutaric dialdehyde for 4 h. The pieces were postfixed in 2% osmium tetroxide for 30 min, dehydrated in graded alcohol, transferred to propylene oxide, and gradually embedded in blocks of Epon 812 resin for 2 days at 60 °C. Eighty-nanometer sections were collected using a diamond histoknife (Diatome) on an Ultracut E microtome (Leica) and then mounted on a copper mesh and stained with uranyl acetate and lead nitrate. The ultrastructural changes in mitochondria was identified by a transmission electron microscope (Hitachi H-7700).

Sciatic nerves were dissected and fixed overnight at 4°C in 2% glutaraldehyde in 0.2 M phosphate buffer. Nerves were then postfixed in 2% osmium tetroxide for 1.5 h at 4°C, and incubated in 2% uranyl acetate for 45 min at 4°C. Nerves were dehydrated in an ethanol series before being incubated with propylene oxide and embedded in epoxy resin. Ultrathin sections (70 nm) were cut with a diamond knife (Diatome), collected onto formvar-coated slot grids and visualised using a transmission electron microscope (T12 Tecnai Spirit, FEI) using a Morada camera and iTEM software (Olympus SIS). Semithin sections were cut using a diamond Histo knife (Diatome) at 0.2 μm, dried and stained with 1% Toluidine Blue in 2% Borax at 75°C for 2 min. Dried sections were mounted with DPX (Sigma-Aldrich) and representative images of the entire nerves were acquired using a wide-field microscope (Zeiss Axio Scope.A1).