Quantifying Sciatic Nerve Ultrastructure

At 26 weeks of age, sciatic nerves were dissected and fixed in 2.5% glutaraldehyde (Polisciences Inc., USA). Then, samples were post-fixed in 1% osmium tetroxide and embedded in epoxy resin (Sigma-Aldrich). For nerve fiber analysis, semithin (0.5 μm) transverse sections were stained with toluidine blue (Sigma-Aldrich), and the area and density of individual fibers were quantified using ImageJ software (NIH, USA). For ultrastructural analysis of the fibers, ultrathin sections were stained with 5% uranyl acetate and 3% lead citrate (Sigma-Aldrich) and examined with a Phillips Morgani electron microscope. G-ratio was analyzed as previously described [34 (link)], evaluating small caliber fibers (< 6 μm diameter) separately from large fibers (≥ 6 μm diameter). Mitochondrial area and density were quantified from electron microscopy images from sciatic nerves, using ImageJ software. Samples from six animals per experimental group were evaluated.