Immunoblots with synaptosomal, cytosolic, and membrane fractions were probed with an affinity-purified rabbit polyclonal antibody against hα-syn (72-10, 1:5000) (Masliah et al., 2000) or with primary antibodies against synaptophysin (1:500, Chemicon, Temecula, CA) or actin (1:1000, Chemicon).
Immunocytochemical analysis of vibratome sections were probed with anti-ha-syn-specific antibody (72-10, 1:500). To analyze the effects of the immunization in glial cell activation, sections were labeled with a mouse monoclonal antibody against the astroglial marker GFAP (1:500, Chemicon) or a rabbit polyclonal antibody against the microglial marker Iba-1 (1:1000, Wako Chemicals, Richmond, VA).
Double-immunocytochemical analysis was performed to determine the effects of vaccination on nerve terminal density and hα-syn accumulation in synapses. Vibratome sections were double-labeled with rabbit polyclonal antibodies against ha-syn (72-10, 1:5000) detected with Tyramide Red (1:2000, Roche, Switzerland) and with the mouse monoclonal antibody against synaptophysin (1:15, Chemicon), detected with a horse anti-mouse IgG FITC-tagged secondary antibody (1:75, Vector). Control experiments were performed where sections were immunolabeled with an antibody against ma-syn detected with Tyramide Red (1:2000, Roche), or with a rabbit polyclonal antibody against b-syn (1:1000, Chemicon), detected with FITC-tagged goat anti-rabbit secondary antibody (1:75, Vector).