Supplemental Material Neuroinflammation and α-Synuclein Dysfunction Potentiate Each Other Driving Chronic Progression of Parkinson’s Disease

Immunostaining and double-label immunofluorescence were performed as described previously using the following primary antibodies: SYN211 (1:500; Millipore), nSYN514 (1:150; Santa Cruz), and antibodies against a neuron-specific nuclear protein (Neu-N; 1:2000; Chemicon), tyrosine hydroxylase (TH; 1:1000; Sigma), or ionized calciumbinding adaptor molecule 1 (Iba-1; 1:500; Wako Chemicals). Briefly, the 30 μm paraformaldehyde-fixed floating brain sections were blocked with appropriate normal serum followed by incubation overnight at 4°C with primary antibodies. The bound primary antibodies were visualized by incubation with an appropriate biotinylated secondary antibody, followed by the Vectastain ABC reagents and color development with 3,3′diaminobenzidine (DAB). For double-label immunohistochemical staining, the 30 μm frozen brain sections were incubated with 99% formic acid for 5 min for antigen retrieval, followed by blocking with 5% horse serum/1% BSA. These brain sections were first stained with the antiNeu-N antibody using DAB as a chromogen and nickel sulfate as an intensifying agent (dark blue) followed with nSYN514 antibody (specific for nitrated human α-syn) using DAB as a chromogen (brown). Images were recorded with a CCD camera and the MetaMorph software (Gao et al. 2003; Giasson et al. 2000). Double-label immunofluorescence was performed by staining brain sections with antibodies against Neu-N and TH, anti-TH antibody combined with SNY211, or anti-Iba1 antibody combined with antiTH antibody, followed by incubation with Alexa-488 (green) and Alexa-594 (red) conjugated secondary antibodies (1:1000). After mounting the sections onto glass slides with the prolong antifade reagents, fluorescent images were obtained with a Zeiss LSM 510 NLO laser scanning confocal microscope fitted with an Argon ion laser (488 nm) and a HeNe laser (543 nm) and recorded with the Zeiss LSM510 software.