Immunohistochemical Analysis of Synuclein Pathology

The procedure for immunohistochemical analysis has been described elsewhere^{60 (link),93 (link)}. Briefly, blind-coded sagittal sections were incubated with the following primary antibodies; anti-α-synuclein(Syn1, #610787, BD bioscience, San Diego, CA, 1:250), anti-α-synuclein (Syn211, # 36-008), anti-NeuN(#MAB377, 1:1000), anti-GFAP (#MAB3402, 1:1000), anti-TH (# AB152, Millipore, County Cork, Ireland, 1:1000), and anti-Iba1 (#019-19741, Wako, Richmond, VA, 1:1000). To detect protease K (PK) resistant α-synuclein aggregates, the sections were pretreated with PK (10 µg/ml) for 8 minutes prior to incubating with anti-α-synuclein antibody^{94 (link)}. After overnight incubation at 4 °C, the sections were incubated with biotinylated secondary antibodies and subsequently detected utilizing an ABC staining kit (both from Vector Laboratories, Burlingame, PA). All sections were imaged by an Olympus BX41 microscope, and the immunoreactivity levels were determined by utilizing ImageJ (NIH). To determine α-synuclein pathology, neurodegeneration, microgliosis, and astrogliosis, the optical density of α-synuclein and GFAP per field (230 mm×184 mm) and the numbers of α-synuclein-positive, NeuN-positive, TH-positive fibers, Iba1-positive cells per indicated field were analyzed using Image Quant 1.43 program (NIH).