Anti iba1 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).

Mice were sacrificed and their brains were immediately removed from the skulls and immersed in formalin. After 24 hours, brains were transferred to a 20% alcohol solution. A 3-mm thick transaxial block, centered at the irradiation site (~3 mm behind the bregma), was obtained from each brain. The blocks were then processed through graded alcohols and embedded in paraffin. All paraffin-fixed blocks were sectioned from the center, at a thickness of five microns. Tissue sections were stained with hematoxylin and eosin (H&E) according to standard protocols.
To measure levels of activated microglia, 5-micron thick tissue sections were immunostained using a rabbit monoclonal anti-IBA-1 antibody (1:1000; Abcam, Cambridge, MA USA), followed by incubation with SuperPicture Polymer Detection Kit, HRP (Life Technologies, Frederick, MD, USA). Slides were viewed with a Hamamatsu NanoZoomer 2.0-HT whole slide imaging system (Hamamatsu Photonics, Bridgewater Township, NJ USA). All histologic and immunohistochemical analyses were performed by a board-certified neuropathologist (S.D.).

Microglia were isolated from primary mixed glial cell cultures prepared from newborn C57BL/6J mice (Guangdong Medical Lab Animal Center, China) on day 10 by shaking the flasks overnight at 300 rpm on a rotary shaker at 37°C. Purified microglia were resuspended and cultured in complete medium containing 1% microglia growth supplement (ScienCell, USA) for 2–3 days. The purity of the cultures was almost 100%, as determined by immunostaining with an anti-Iba1 antibody (Abcam, USA). The protocols for the animal experiments were approved by the Animal Experiment Committee of Guangzhou Medical University. Mouse EOC 2 microglial cells were obtained from the American Type Culture Collection (http://www.ATCC.org) and were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco-BRL) containing 10% fetal bovine serum (FBS; Gibco-BRL).