Erpendicular to the nerve’s long axis, were cut on an ultramicrotome and picked up on an osmium-coated glass slide. Back-scattered images were obtained on a scanning electron microscope (Hitachi SU8010) with a working distance of 2 mm, 1.5 kV accelerating voltage, scan speed of 40 or 80 s, and a typical pixel size of 1.65 nm at x30,000 magnification, and were analysed with ImageJ (FIJI). Node length (assessed from the number of sections containing the node) and mean axon diameter at the node (assessed as axon perimeter/p) were measured (uncorrected for tissue shrinkage during fixation).Tracer injections and node labellingFour male 80 week old rats were anaesthetized with isoflurane and killed by cervical dislocation in accordance with United Kingdom animal experimentation regulations. After decapitation the brain was carefully dissected from the skull and 1 mm thick coronal slices containing the corpus callosum were obtained from the forebrain (from 4 to 8 mm rostral of the olfactory bulb) using a tissue cutter block. A 10 solution of tetramethylrhodamine dextran (MW 3000, Invitrogen, Paisley, UK) was iontophoretically injected into the cortical grey matter. Thereafter slices were incubated in oxygenated aCSF containing (in mM) 124 NaCl, 26 NaHCO3, 1 NaH2PO4, 2.5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, bubbled with 95 O2/5 CO2 for 2 hr at room temperature to allow for diffusion of the tracer. After incubation slices were immersion fixed in PFA and resliced at 8000 mm for subsequent immunohistochemical labelling of nodal (NaV1.6) and paranodal (Caspr) marker proteins.Immunohistochemistry and confocal microscopyOptic nerves and 4 mm thick coronal sections of fronto-parietal (motor) cortex (from 4 to 8 mm rostral of the olfactory bulb) from brains of 4 male (80 week old) Sprague-Dawley rats were either perfusion or immersion-fixed in 4 paraformaldehyde in PBS. Fixed tissue was then cut into 50 mm slices using a Leica vibratome VT1200S or, for NaV1.6 density experiments, cut into 10 mm sections using a cryostat. Slices were blocked and permeabilised in 10 horse serum and 0.5 Triton X-100 in PBS. Immunofluorescence labelling was performed over 3 days with the following primary antibodies: rabbit anti-NaV1.Zoliflodacin 6 (Alomone, 1:500); mouse anti-Caspr clone K65/35 (Neuromab, UC Davies, 1:100).Dp44mT Slices were then washed extensively (3 20 min) and incubated overnight with secondary antibodies: anti-rabbit AlexaFluor488 (Invitrogen, 1:500), anti-mouse Dy-Light 647 (Jackson Immunoresearch, 1:500).PMID:23563799 Slices were then washed 3 10 min in PBS and mounted with Dako Fluorescent Mounting Medium. Slices were viewed using an LSM700 or LSM780 confocal microscope using a 63x (NA 1.4) oil immersion lens, and images were acquired with LSM software with the pinhole set to 1 Airy unit for the Caspr signal, resulting in an optical slice of 0.8 mm. Pixel size was 39.7 nm for Figure 1A and 99.2 nm for Figure 1I and 52.7 nm for node measurements in Figure 2 and 263.6 nm for internode measurements in Figure 2.Image analysisFor node length analysis, confocal images were analysed using ImageJ software. Images were background subtracted and only nodes that lay approximately parallel to the plane of section (i.e. displayed nodal NaV1.6 labelling with flanking Caspr-labelled paranodes all within a single 0.8 mm optical slice) were selected. Measuring the angle of the axon to the plane of the slice for a subset of 10 randomly chosen axons showed that the apparent node length measure.
