Toluidine blue staining and electron microscopy preparation

Mice were perfused with 4% PFA, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4. Spinal cords were removed, and lumbar regions were processed by the Cleveland Clinic Electron Microscopy Core. Tissue was postfixed in 1% osmium tetroxide in water, stained with 1% uranyl acetate in maleate buffer (pH 5.1), and dehydrated with ethanol and propylene oxide before being embedded in Pure Eponate 12 resin (Ted Pella Inc., Redding, CA). Semithin 1-μm sections were cut with a diamond knife and stained with toluidine blue for analysis by light microscopy. Ultrathin 85-nm sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope.