Bovine serum

For immunostaining analysis, non-specific binding was blocked with 3% bovine serum (Serotec, UK), and permeabilized with 0.1% Triton X-100 in 1% BSA-PBS for 30 min. The sections were incubated at 4°C overnight with polyclonal rabbit anti-TH (1:500; Millipore, USA), monoclonal anti-α-synuclein (1 : 2000, a gift from Prof. Shun Yu), anti-CD11b (1:500; eBioscience, USA), p-NF-кB/p65 (1:1000; Chemicon, USA), polyclonal rabbit anti-Nurr1/NR4A2 (1:500; Proteintech, China) and then incubated with corresponding secondary antibodies at room temperature for 2h. The nucleus was stained by Hoechst 33342 (1µg/ml, Sigma-Aldrich, USA). We cut 100 pieces in total from anterior to posterior of the SN, and then chose six sections from each 20 pieces. Each section in the different groups was at the same level of the SN or striatum. THimmunoreactive (TH-ir) neurons were stereologically counted from anterior to posterior of the SN.
The immunofluorescent intensity of TH in the striatum was determined based on optical density analysis by Image-Pro Plus software. Quantitative analysis of positive staining (the expression of p-NF-кB-p65, IL-1β, TNFα and Nurr1 on CD11b + microglia) was determined based on number of double positive cells/mm 2 by Image-Pro Plus software. Images were taken from the same location in all animals.

On day 22 p.i., mice were perfused and fixed with saline and 4% (w/v) buffered paraformaldehyde. The brains and spinal cords (lower thoracic-lumbar) were removed and sliced (10 μm). The pathological changes were detected by hematoxylin/eosin (H&E) staining. For immunohistochemistry, non-specific binding was blocked with 3% bovine serum (Serotec, UK) in 0.3% Triton X-100/PBS for 30 minutes at room temperature (RT). The sections were incubated with anti-CD4 (1 : 1000; eBioscience), anti-CD68 (1 : 1000; eBioscience), anti-CCL2 (1:500; Abcam UK), and anti-CCL5 (1 : 500; Abcam UK) at 4°C overnight. The sections were incubated further with corresponding secondary antibodies at RT for 2 h. The negative controls were treated similarly, but the primary antibodies were omitted.