Immunohistochemistry was performed using previously established methods [36 (link), 37 (link)]. Briefly, sections were blocked [10% normal goat serum, 0.5% Triton-X-100 in Tris buffered saline (TBS, pH 7.4)] and incubated in primary antibodies, anti-α-Syn (clone 42, 1:500 - 610787, BD Biosciences, San Jose, CA), anti-phospho α-Syn S129 (1:300 - ab-59264, Abcam, Cambridge, UK), anti-tyrosine hydroxylase (1:4000 - MAB318, Chemicon Temecula, CA) and anti-Iba1 (1:500 - 019-19741 Wako Chemicals, Richmond, VA) overnight at room temperature (RT). Primary antibodies were detected in a 2 hr incubation at RT with secondary antibodies coupled to fluorochromes Alexa 488 or 555 (Life Technologies-Molecular Probes, Grand Island, NY) and counterstained with 4',6'-diamidino-2-phenylindole, dihydrochloride (DAPI, Life Technologies). Alternatively, primaries were treated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) followed by ABC reagent (Vector Laboratories) and exposure to 3'-Diaminobenzidine (DAB: Sigma Aldrich, Saint Louis, MO). Control conditions constituted the deletion of the primary antibody or secondary antibody and the inclusion of relevant isotype specific antibodies and sera instead of the omitted antibodies. Sections probed with α-Syn and phospho α-Syn were counterstained with Hematoxylin.
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