Diamond knife

Tissue samples of the cranial right lung lobe were fixed for 24 h in 2.5% glutaraldehyde in 0.2 M sodium phosphate buffer (pH 7.3) for transmission electron microscopy (TEM) examination in the TEM Unit (Institute of Veterinary Pathology, Laboratory for Animal Model Pathology, University of Zurich). Briefly, specimens were washed in sodium phosphate buffer and fixed in 1% osmium tetroxide in phosphate buffer/distilled water for 60 min. Tissues were dehydrated in ascending concentrations of ethanol, followed by propylene oxide. Specimens were subsequently infiltrated with 50% resin (Epoxy embedding medium, Sigma Aldrich) and incubated overnight. The tissues were then incubated in polyethylene embedding capsules filled with 100% resin for 3 days.
Semi-thin sections (1 µm) were prepared using an ultramicrotome (Reichert-Jung Ultracut, Munich, Germany) with a diamond knife (Diatome, Biel, Switzerland), stained with toluidine blue, and areas of interest for the preparation of ultrathin sections were chosen under the light microscope. Ultrathin sections (90 nm) were prepared with a diamond knife, mounted on copper grids, stained with uranyl acetate and Reynold's lead citrate, and examined with a transmission electron microscope (Philips CM10, FEI, Thermo Fisher Scientific) operating with a Gatan Orius Sc1000 digital camera (Digital Micrograph, Gatan, Pleasanton, CA, USA).