Ultrostainer

Intercostal nerves were incubated for 48 h in 4% PFA: 2.5% glutaraldehyde at 4°C before post-fixation in 1% osmium tetroxide in 0.1 M phosphate buffer for 45 min. Following dehydration through an ascending series of ethanol solutions and propylene oxide, sections were embedded on glass slides in Durcupan resin. Regions to be used for the assessment of myelination were then cut out from a randomly selected section using a scalpel and glued onto a resin block for sectioning. Ultrathin sections (60 nm) were cut and collected on formvar-coated grids (Agar Scientific, UK), stained with uranyl acetate and lead citrate in an LKB Ultrostainer and then quantitatively assessed in a Philips CM12 transmission electron microscope equipped with a Gatan digital camera. Intercostal nerve fibres were measured using ImageJ. For each individual fibre, axon diameters and G-ratios were calculated as previously described (46 (link)).

Liver tissue for transmission electron microscopy was prepared following immersion fixation in 0.1 M PB buffer (pH 7.4, EM-grade) containing 4% paraformaldehyde and 2.5% glutaraldehyde. 1mm tissue blocks were post-fixed in 1% osmium tetroxide in 0.1 M PB for 45 min before dehydration through an ascending series of ethanol solutions and propylene oxide. Tissue blocks were then embedded in Durcupan before ultrathin sections (∼60/70 nm) were cut and collected on formvar-coated grids (Agar Scientific, UK), stained with uranyl acetate and lead citrate in an LKB Ultrostainer and then quantitatively assessed in a Philips CM12 transmission electron microscope (TEM).