Anti th antibody

After separation, the rats’ midbrains were fixed in 4% paraformaldehyde for 24 h at 4 °C, and then processed using the following procedure: alcohol (70%) for 1 h, alcohol (80%) for 1 h, alcohol (90%) for 1 h, alcohol (100%) for 1.5 h, alcohol (100%) for 1.5 h, Xylene for 30 min, dipping wax for 1 h. Then, midbrains were embedded in paraffin and sectioned (3 μm per slice). The immunohistochemistry process was performed with an Ultra-SensitiveTM S-P kit (contains endogenous peroxidase blocking solution, sheep serum, biotin-labeled goat anti-rat secondary antibody, and streptavidin-peroxidase) (MBX Biotechnologies, Fuzhou, China). The dopaminergic neurons were labeled with the anti-TH antibody (Abcam, Cambridge, UK, Diluted with 5% fetal bovine serum), and the microglia was labeled with the anti-IBA-1 antibody (Abcam, Cambridge, UK, Diluted with 5% fetal bovine serum). The experimental procedures were manipulated as previously described [39 (link)]. To determine TH- and IBA-1-positive cell numbers, the cells were respectively quantified by five researchers who had absolutely no hand in the experimental treatments.

Mice were sacrificed to collect the brain and frozen at -80°C. Brain tissues were lysed, and centrifuged at 12000 rpm and 4°C for 20 min to obtain the total protein in the supernatant. The protein concentrations were estimated by the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Rockford, IL, USA). Then, the protein was boiled in sodium dodecyl sulfate (SDS)-loading buffer at 95°C for 10 min to denature. The appropriate amount of protein was run on a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm polyvinylidene fluoride membranes, and the membranes were blocked with 5% fat-free milk at room temperature for 1 h. The membranes were incubated at 4°C overnight with primary antibodies and incubated at room temperature for 1 h with the HRP-conjugated secondary antibody. Finally, an enhanced chemiluminescence reagent (Thermo Scientific Rockford) was used to develop the blots with a gel image system (Bio-Rad, CA, USA), and the analysis is performed by using ImageJ 1.49v (National Institutes of Health, USA, https://imagej.en.softonic.com/). The following antibodies were used: anti-TH antibody (Cat# ab112, Abcam, Cambridge, MA, USA), anti-β-actin antibody (Cat# 8457S, Cell Signaling Technology, Berkeley, CA, USA), anti-α-syn antibody (Cat# ab212184, Abcam), goat anti-rabbit antibody (Cat# ab6721, Abcam),.

Lipopolysaccharide was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). IL-1β, IL-6, TNFα, MCP-1, and ICAM-1 ELISA kits were purchased from Genetimes Technology Inc. (Shanghai, China). Anti-TH antibody, anti-PSD-95 antibody, anti-COX-2 antibody, anti-Claudin-1 antibody, anti-Occludin antibody, and anti-CX43 antibody, anti-HMGB1 antibody, anti-TLR4 antibody, and Cy3-conjugated secondary antibody were the products of Abcam (Cambridge, United Kingdom). Anti-Iba-1 was the product of Wako (Osaka, Japan).