Ecl reagent

For Western blot, mice were perfused transcardially with PBS to remove blood. The brains were collected and different brain regions were dissected on ice. All the samples were stored at −80 °C. The brainstem samples were homogenized in cold RIPA lysis buffer containing proteinase and phosphatase inhibitors.28 (link),36 (link) Equivalent amounts of protein from each group were added to a 4–12% Bis-Tris-polyacrylamide electrophoresis gel. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. We blocked the membranes with 5% skim milk and then incubated them with primary antibodies against TH (1:1000, ab129991, Abcam, Cambridge, MA, USA), Iba-1 (1:1000, 019–19,741, Abcam, Cambridge, MA, USA), CR3 (1:1000, ab128797, Abcam, Cambridge, MA, USA) or GAPDH (1:5000, ab181602, Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were then washed three times with PBST and incubated with horseradish peroxidase-linked anti-rabbit IgG (1:3000) antibody at room temperature for 2 h. The blot signals were detected by ECL reagents (Biological Industries, Cromwell, CT, USA). The densitometry of blots was quantified based on previous report.46 (link) GAPDH was used as an internal control for each blotting. All blots were normalized to GAPDH. Fold changes for each treatment were normalized and are shown as percentages of the control.

Equal amounts of protein were separated by 4–12% Bis-Tris-polyacrylamide electrophoresis gel and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibody Neu-N, PSD-95, tyrosine hydroxylase (TH, EMD Millipore, Temecula, CA, USA), α-synuclein, signal transducers (Abcam, Cambridge, MA,USA) and activators of transcription 1 (STAT1), p-p65, p65, p-IκBα and IκBα (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and followed by horseradish peroxidase-linked anti-rabbit IgG (1:3000) for 2 h at 25 °C. ECL reagents (Biological Industries, Cromwell, CT, USA) were used as a detection system.