Uct ultramicrotome

TEM was used to measure myelin thickness in the CC/EC area. Mice were perfused with ice-cold saline, followed by 4% PFA and 2.5% glutaraldehyde in 0.1 mol/L PBS buffer. The CC/EC tissue near the site of ischemia was microdissected into 1-mm³ blocks. These specimens were immersion-fixed for 24 h in 2% glutaraldehyde. Tissue was then rinsed in PBS and fixed in 1% osmium tetroxide in 0.1 M PBS for 45 min. Samples were dehydrated in increasing concentrations of acetone and embedded in Araldite resin. Ultrathin 60-nm sections were cut on a Leica UCT ultramicrotome with a diamond knife (Diatome, Wetzlar, Germany), stained with uranyl acetate and lead citrate, and viewed using a JEM1400 TEM (JEOL, Akishima, Japan). Two sections from each animal were analyzed. Three to 5 images (600 μm² each) were acquired in randomly selected areas within the CC/EC from each section at a magnification of 50,000× and analyzed with Image J by an investigator blinded to experimental groups. At least 50 random axons per animal were analyzed by tracing the axonal circumference and the whole fiber circumference in a blinded fashion. G-ratios were calculated as the ratio of the inner axonal diameter to the total outer diameter (axonal diameter + total myelin sheath thickness).

Male mice between 10 and 13 wk of age were perfused with 3% glutaraldehyde and 2% PFA in 0.1 M phosphate buffer (pH 7.4) with 0.7% (wt/vol) NaCl, and spinal cords were dissected and postfixed overnight. Specimens were rinsed in 0.1M cacodylate, postfixed in 1% osmium tetroxide (Agar Scientific), dehydrated, and embedded in Durcupan resin (Sigma). Semithin sections (750 nm) were cut on a Leica UCT ultramicrotome using a Histo knife (Diatome) and mounted on microscope slides. Semithin sections were incubated in a 1% toluidine blue solution for 43 s at 60 °C and rinsed with water. Brightfield images of healthy vWM tissue derived from lesioned spinal cords were acquired on a Leica DM5500 microscope. The number of myelinated axons were counted by a blinded observer on three to four sections per animal as above.

Animals were fixed with 2% glutaraldehyde in 0.1 M Nacacodylate buffer, pH 7.3, for 2 h at room temp, rinsed in buffer, and post-fixed with 1% OsO₄ in a buffer for 1 h at room temp. The post-fixed samples were then rinsed with water, stained en bloc with 5% uranyl acetate in water, dehydrated in ethanol and propylene oxide, and embedded in Eponate 12 resin (Ted Pella, Inc., Redding, CA). 50 nm sections were cut with a Leica UCT ultramicrotome using a Diatome diamond knife, picked up on slot grids with Pioloform films, stained with uranyl acetate and Sato’s lead, and examined with an FEI T12 TEM at 120 kV equipped with a Gatan Ultrascan 4k × 4k camera.