Mouse monoclonal anti synaptophysin

Seven days before the end of behavioral observations, 5% Fluoro-Gold solution (Sigma-Aldrich) was injected into the tibial anterior and gastrocnemius muscles of right and left hindlimbs (2.5 μL/site). Spinal cord sections at the L2-L6 level were prepared as described above for the 5-HT immunohistochemistry. Sections were post-fixed in 4% paraformaldehyde, immunostained with the mouse monoclonal anti-synaptophysin (1:500; Sigma-Aldrich) and rabbit monoclonal anti-Fluoro-Gold (1:500; Fluorochrome, Denver, CO) antibodies at 4°C for 24 h, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (1:400; Life Technologies) and Alexa Fluor-Cy5-conjugated anti-rabbit IgG (1:50; Life Technologies) secondary antibodies, and counterstained with DAPI. Images were obtained using a fluorescence microscope (BZ-ZX700/BZ-X710; Keyence) and quantified using Image J (National Institutes of Health). The number of Fluoro-Gold–positive motor neurons was counted. Synaptophysin-positive puncta on Fluoro-Gold–positive motor neuron cell bodies were quantified. All slices containing Fluoro-Gold–positive neurons were quantified.