H 7100 electron microscope

The lateral corpus callosum of mice was rapidly dissected and immersed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 overnight at 4 °C. Samples were processed as previously reported^{34 (link),35 (link)}. After washing three times with the same buffer, the samples were postfixed in 1% osmium tetroxide buffered with cacodylate for 1 h at room temperature, dehydrated through a graded series of ethanol and embedded in Spurr’s resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Hitachi H-7100 electron microscope equipped with a Gatan 832 digital camera (Gatan, 86 Inc.). Nonoverlapping EM images of the coronal-sectioned corpus callosum were analyzed to determine the g-ratio as previously reported^{36 (link)}. Images were processed with ImageJ software (https://imagej.nih.gov/ij/) with a plug-in (https://gratio.efil.de) that allowed randomly selected axons to be analyzed. The outer border (used to calculate myelin area) and inner border (used to calculate axon area) of myelin sheath were manually marked, and the g-ratio was calculated as the axonal diameter divided by the myelinated fiber diameter. A total of 300 axons from 3 different images from the control mice and 600 axons from 6 different images from the PD mice were analyzed.

Three days following cotransfection of the plasmids encoding the MERS-CoV structural proteins, cells were fixed with 4% glutaraldehyde in 0.1 M phosphate buffer for 24 h at 4 °C and post-fixed 1% aqueous osmium tetroxide diluted in the same phosphate buffer at room temperature for 1 h. After washing, the fixed cells were dehydrated in a graded ethanol series and embedded in a Polybed 812-Araldite mixture (EMS, Hatfield, PA). Semi-thin sections of 1 μm were obtained using an ultramicrotome (Ultracut E, Leica-Reichert Jung, Wetzlar, Germany) and stained with toluidine blue for correlative light microscopy. Ultrathin sections were cut at 70 nm, collected on copper grids (200 meshes) and stained with uranyl acetate and lead citrate. Images were examined in a Hitachi H-7100 electron microscope equipped with a Gatan 832 digital camera (Gatan, Inc.).