Anti α syn antibody

For immunohistochemistry (IHC), the brains were separated in PFA solution at 4°C overnight and embedded with 3% agarose in PBS. Coronal sections were gathered using VT1000 vibratome (Leica Biosystems, Wetzlar, Germany) at a thickness of 30 μm throughout the brain, including the SNpc and striatum and every 3 sections were analyzed. 3% H₂O₂ was used to block endogenous peroxidase activity for 10 min at room temperature and sections were blocked with 10% donkey serum for 10 min. For immunofluorescence (IF), sections were blocked with 10% donkey serum for 30 min. Then, the sections were incubated with primary antibody. The sections were incubated with an HRP-conjugated anti-rabbit antibody, stained using the DAB kit (Vector Labs, Carlsbad, CA, USA), and were incubated overnight at 4°C. Fluorescein was combined with a fluorescent secondary antibody. Images were gathered using a fluorescence microscope (Olympus, Tokyo, Japan). The quantifications were performed using ImageJ. The following antibodies were used: anti-Iba1 antibody (Cat# ab5076, Abcam), anti-GFAP antibody (G3893, Sigma-Aldrich, St Louis, MO, USA), anti-α-syn antibody (Cat# ab212184, Abcam), anti-pSer129-α-syn antibody (Cat# ab51253, Abcam), Goat anti-rabbit antibody (Cat# ab6721, Abcam), donkey anti-mouse antibody (Cat# A21203, Invitrogen, Carlsbad, CA, USA), donkey anti-goat antibody (Cat# A32758, Invitrogen).

The protein was isolated from SH‐SY5Y cells using RIPA buffer containing protease inhibitor cocktail (Sigma), and aliquots containing 40 μg of total protein were separated into 12% SDS gel and transferred onto 0.45 μm PVDF. After blocking in 5% nonfat milk, the membranes were then incubated overnight with the following primary antibodies: anti‐α‐syn antibody (Abcam) and anti‐β‐actin antibody (Abcam). The secondary antibodies conjugated with horseradish peroxidase were used. The detection of the proteins was carried out by BCA Protein Assay Kit (Thermo) according to the manufacturer's instruction. The proteins were visualized by ECL chemiluminescence and exposed to X‐ray film. All experiments were performed in duplicate and repeated three times.

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),.