Histo hi 4317

Mouse sciatic nerves were fixed for 20 min in situ with 4% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), then removed and post-fixed overnight in the same buffer. After washing 30 min in 0.2 M PBS phosphate buffer, samples were incubated with 2% osmic acid in 0.1 M phosphate buffer for 90 min at RT. They were then washed in 0.2 M PBS phosphate buffer, dehydrated using ethanol gradient solutions and embedded in epoxy resin. To obtain electron microscopy images, ultrathin (70 nm) cross-sections were cut and stained with 1% uranylacetate solution and lead-citrate and analyzed using a HITACHI H7100 electron microscope. Semi-thin cross-sections (0.7 μm) were cut using a microtome (Leica, RM 2155, Wetzlar, Germany) with a diamond knife (Histo HI 4317, Diatome, Hatfield, UK). Sections were stained with blue from toluidine and imaged using an AxioScan slide scanner (Zeiss, Nantes, France). G-ratio was determined using the GRatioCalculator plugin of Image J. At least 200 fibers were analysed per animal.

Nerves were removed and fixed in 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde in 0.1 M PB, contrasted with osmium tetroxide, and Epon embedded. Semithin cross-sections (0.5 µm) were cut using a microtome (Leica, UC7) with a diamond knife (Histo HI 4317, Diatome). Sections were stained with Azur II-methylene blue for 1 min at 60 °C. Light microscopic images were captured with the Axio Imager Z1 (Zeiss) in ×100 maginification. Analysis was performed on total nerve cross-section. For EM of nerves, ultrathin (50–70 nm) cross-sections were stained with 1% uranylacetate solution and lead citrate^{60 (link)} and analyzed using a Zeiss EM900 or LEO912. For quantification of histological parameters as the number of unmyelinated fibers, Schwann cell nuclei, and the g-ratio (calculated by determination of the axon area and the area of the fibre including the myelin sheath and subsequent calculation of the diameter of a hypothetical circular area, and subsequent division of the axon diameter by the fibre diameter), at least 20 random taken pictures were captured at a magnification of ×3000.