To extract tissue for cryo-sectioning, animals were culled by overdose of pentobarbital delivered via intraperitoneal injection, followed by transcardial perfusion with 15ml sterile 0.9% w/v saline, then 20ml 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB). Dissected tissues were post-fixed in 4% PFA for varied duration depending on tissue, as determined previously in our laboratory (Dawes et al., 2018 (link)): L4 DRG – 2hrs (room temperature, RT), spinal cord – 24hrs (4°C), glabrous skin – 0.5hrs (RT). For analysis of epidermal nerve fibres, skin was dissected from the glabrous region of the hind paw proximal to the most proximal touch dome, as depicted in (Fig. 6A). After fixation, tissue was transferred to 30% sucrose in 0.1M PB at 4°C for at least 24 hours. Subsequently, tissue was embedded in OCT medium (TissueTek), rapidly frozen with liquid nitrogen, and stored at -80°C prior to cryo-sectioning. Sections were slide-mounted on Thermo Scientific™ SuperFrost Plus™ slides – with sections cut using a Leica cryostat, at tissue-dependent thickness: DRG – 10μm; spinal cord – 20μm, skin – 14μm.