Superfrost gold plus glass slides

Mice were anaesthetised with pentobarbitol (30 mg/kg dose, Dolethal), and the chest cavity opened to reveal the heart. The right atrium was severed and 10 mL ice cold 1 × phosphate buffered saline (PBS) was perfused through the left ventricle, followed by 10 mL 4% paraformaldehyde (PFA; Alfa Aesar). The lumbar spinal cord was then dissected and incubated for a further 3–4 h (h) in 4% PFA before being incubated in sucrose 30% w/v for up to 72 h at 4°C until sunk. Prior to cryo-embedding, the spinal cord was cut at approximately Lumbar segment 1 (L1), which was determined as being approximately 2 mm rostral of the lumbar enlargement, and by examining the distribution of the ventral roots. Thus, the first cryosections were taken at approximately Lumbar segment 1–2 (L1-2). Tissue was then cryo-embedded in OCT compound and stored at − 80°C. Cryosections at 20 μm thickness were obtained using a Leica CM1860 cryostat and adhered to Superfrost Gold Plus glass slides (VWR). For experiments requiring total internal reflection fluorescence (TIRF) microscopy, 10 μm thick sections were adhered to an Argon Plasma cleaned coverslip (1.0 thickness).

Mice were anaesthetised with pentobarbitol (30 mg/kg dose; Dolethal), and the chest cavity opened to reveal the heart. The right atrium was severed and 10 ml ice cold 1 × phosphate buffered saline (PBS) was perfused through the left ventricle, followed by 10 ml 4% paraformaldehyde (PFA; Alfa Aesar). The lumbar spinal cord was then dissected and incubated for a further 3–4 h in 4% PFA before being incubated in sucrose 30% w/v for 24–72 h at 4 °C until sunk. Tissue was then cryo-embedded in OCT compound and stored at − 80 °C. Cryosections were obtained using a Leica CM1860 cryostat at 20 μm thickness and adhered to Superfrost Gold Plus glass slides (VWR).