Tissue Processing for Ultrastructural Analysis

Mice were deeply anesthetized with 10 % chloral hydrate and killed by transcardial perfusion with 1× PBS followed by 4 % paraformaldehyde in 1× PBS. Tissues were post-fixed in the same fixative overnight. Sciatic nerves were cryoprotected in 30 % sucrose in 1× PBS, and sectioned at 20 μm. Sciatic nerves were sectioned longitudinally using a Leica cryostat (model CM3050S; Leica Microsystems). For semithin sections, nerves, DRG, and spinal cords were postfixed in 4 % paraformaldehyde/2 % glutaraldehyde followed by OsO₄ and embedded in Epon. Cross sections, at 1 μm thickness, were stained for toluidine blue and examined under light microscopy. For ultra-structural analyses, spinal cord and nerves were processed as described previously [24 (link)]. Briefly, 70 nm thin sections were obtained and stained for citrate/uranyl acetate. Electron micrographs were acquired using Zeiss Libra transmission electron microscope.