G3231

P73 (n = 4) WT and SOD1^G93A mice were culled, and sciatic nerves were immediately dissected, post-fixed in a 4% PFA solution in PBS overnight at 4 °C, cryopreserved in a 30% sucrose solution in PBS for 2 d at 4 °C, and finally frozen in OCT (Agar Scientific, AGR1180). 30 µm longitudinal cryosections of sciatic nerves were directly mounted on SuperFrost Plus slides (VWR, 631–0108), and a hydrophobic barrier pen (Vector Laboratories, H-4001) was then applied to the slides surrounding the sectioned tissue. PBS rehydrated tissue was then blocked using 10% normal horse serum in 0.2% Triton X-100 in PBS for ~ 1 h and then primary antibodies (see Additional file 2: Table S3) specific for S100 (Merck, S2532, 1:200), TUJ1 (Synaptic systems, 302306, 1:500), TrkB (Millipore, 07–225, 1:250) and p75^NTR (Promega, G3231, 1:500) were applied overnight at room temperature. After multiple PBS washes, the secondary antibodies (see Additional file 2: Table S4) in PBS were applied for 2–3 h, followed by multiple washes and then mounted with Mowiol. Slides were dried and imaged as described above. A minimum of six sciatic nerve sections were imaged per condition. Mean fluorescence was measured by applying a TUJ1 and S100 mask to the TrkB or p75^NTR regions, and the mean fluorescence per animal was assessed by averaging all the individual data points.