Dylight 647

Mice were anesthetized with choral hydrate and perfused with saline followed by 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS). The brains were removed, fixed in 4% paraformaldehyde overnight, and subjected to dehydration in increasing saccharin solutions (20–30%) at 4^ºC. The frozen coronal slices of 20 μm thicknesses were prepared and stored at -20^ºC in 20% ethanediol PBS solution containing 20% saccharin. Brain sections were incubated in 3% normal goat serum and 0.2% Triton-X for 1h. Then they were incubated in rabbit anti-Parvalbumin (Swant, Bellinzona) or mouse anti–corticotropin-releasing factor (CRF; Abcam) antibody overnight at 4ºC. Slices were rinsed in PBS then incubated in donkey anti-rabbit Cy3, Alex488, DyLight 647, or donkey anti-mouse Cy3 (Jackson Immunoresearch) for 1h and 4',6-diamidino-2-phenylindole (DAPI) for 10min, then mounted after rinsing. Images were acquired on a microscope using a 20× air objective, a 40× objective (IX-83; Olympus) or a 63× oil immersion objective (Zeiss 510; Carl Zeiss Jena).

Optic 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 (8–10 week old) Sprague-Dawley rats were either perfusion or immersion-fixed in 4% paraformaldehyde in PBS. Fixed tissue was then cut into 50 µm slices using a Leica vibratome VT1200S or, for Na_V1.6 density experiments, cut into 10 µm 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-Na_V1.6 (Alomone, 1:500); mouse anti-Caspr clone K65/35 (Neuromab, UC Davies, 1:100). 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). 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 µm. Pixel size was 39.7 nm for Figure 1A–H 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.