Spurr s resin

Electron microscopy used in this study followed a previously described method [28 (link)]. Briefly, C6/36 cells seeded on the dish were immediately fixed with a mixture of 2% (v/v) glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. After cells were postfixed in 1% (w/v) osmium tetroxide in 0.1 M cacodylate buffer for 2 h at room temperature, they were washed with 0.2 M cacodylate buffer three times. Again, cells were washed with 0.2 M cacodylate buffer three times and then dehydrated through an ascending graded series of ethanol. Cells were embedded in situ with Spurr's resin (Electron Microscopy Science, Hatfield, PA, USA), followed by polymerization at 70°C for 72 h. Trimmed blocks were sectioned with an ultramicrotome (Reichert Ultracut R, Leica, Vienna, Austria), and the ultrathin sections were stained with saturated uranyl acetate in 50% ethanol and 0.08% lead citrate in sequence. Selected images were observed and photographed under a transmission electron microscope (JEOL JEM-1230, Tokyo, Japan) at 100 kV.

Mice were killed with 150 mg/kg pentobarbital i.p. (Esconarkon; Streuli Pharma AG) and sciatic nerves or spinal cords were fixed in situ (sciatic nerves) or by perfusion (spinal cords) with 4% paraformaldehyde (PFA) and 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Fixed tissues were post-fixed in 2% osmium tetroxide, dehydrated through a graded acetone series^{40 (link)}, and embedded in Spurr’s resin (Electron Microscopy Sciences). Ultrathin sections (70-nm thick) were cut. G-ratio (diameter of axon: diameter of axon+myelin) analyses were done at 12 mm distal to the lesion site for sciatic nerves and in the lesion site for spinal cords (g ratio average of 60–250 axons calculated per animal). To delineate the lesion site in the spinal cord and identify remyelinated axons, the g-ratio was calculated. Axons with a g-ratio above or equal to 0.835 were classified as remyelinated axons. This g-ratio threshold value was defined after calculating the g-ratio of axons in non-lesioned mouse spinal cords, where axons with the thinnest myelin sheath had a g-ratio under or equal to 0.83. No contrasting reagent was applied. Images were acquired using a Philips CM 100 BIOTWIN equipped with a Morada side-mounted digital camera (Olympus).

For light microscopy, stem segments embedded in low viscosity (Spurr's) resin (Electron Microscopy Sciences) were cut into 1-µm-thick sections with a microtome and stained with toluidine blue [41] . Presence of lignin was visualized by staining the sections with phloroglucinol-HCl or using a UV fluorescence microscope [7] (link). Secondary wall cellulose was stained by incubating sections with 0.01% Calcofluor White [42] (link). Under the conditions used, only secondary walls exhibited brilliant fluorescence. Xylan was detected by using the monoclonal LM10 antibody against xylan and fluorescein isothiocyanate-conjugated goat anti-rat secondary antibodies [43] (link).