Immunohistochemistry of Sciatic Nerve

Animals were sacrificed after the behavioral test for immunohistochemistry. The sciatic nerve was collected according to a previously described method [20 (link)]. The sciatic nerves were dissected and immediately immersed in a solution consisting of 4% paraformaldehyde in 100 mM PBS overnight. For cryoprotection, fixed tissue was immersed in a solution consisting of 30% sucrose in PBS. Longitudinal sections 20 μm thick were cut using a cryostat (Leica, Nussloch, Germany). The tissue slides were incubated overnight at 4°C with primary antibody to neurofilament protein (NF-200; Sigma). After washing, the slides were incubated with secondary FITC anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature. Tissues were mounted, and images were acquired using a Nikon Eclipse 50i (Nikon Inc., Melville, NY, USA) fluorescence microscope (100× to 400×). Analyses of individual sections used in the final quantification were performed using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD, USA).