Flex slides

At the appropriate time point, animals were deeply anesthetized with sodium pentobarbitone (60 mg/kg, intraperitoneally). When all reflexes were absent, mice were transcardially perfused with 10 mL 10 mM PBS followed by 20 mL 4% w/v paraformaldehyde (PFA) in PBS. The T12-L1 vertebrae, containing the L2-L5 lumbar spinal cord segment, were dissected and post-fixed in 4% PFA overnight at 4°C, then stored in PBS with 0.01% w/v sodium azide. Cervical vertebrae, from the subset of animals described above, were collected in the same manner. All samples were incubated in rapid decalcification solution (5% v/v nitric acid with 0.05% w/v urea) for 2 hours at room temperature, then washed thoroughly with distilled water. Decalcified samples were dehydrated and embedded in paraffin wax using an automated tissue processor (Leica Biosystems ASP200-S, Melbourne, Australia). Sections (4 μm) were cut on a microtome (Microm HM325, Walldorf, Germany), mounted on Flex slides (Dako), dried at 37°C overnight and then stored at room temperature. Prior to immunohistochemistry, paraffin-embedded sections were heated to 60°C for 10 minutes for efficient paraffin removal, dewaxed in xylene and rehydrated through a series of graded ethanols to distilled water.