Ab218011

Brain tissue was dissected quickly from the corpus callosum after deep anaesthesia. The samples were processed for western blotting analysis as previously described.²² The primary antibodies used were: rabbit monoclonal anti‐MBP (1:1000; ab218011; Abcam), rabbit polyclonal anti‐Olig2 (1:1000; AB9610; Millipore), and mouse monoclonal anti‐β‐actin (1:4000; 30101ES60; Yeasen). After incubation with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit or goat anti‐mouse secondary antibody (1:5000; ZB5301/SA00001‐1; Zhongshanjinqiao/Proteintech), and bands were detected using enhanced chemiluminescence reagent (ECL, Pierce) and quantified using ImageJ software.

Following deparaffinization and hydration, sections were selected randomly from the lumbar spinal cord and hippocampus. All sections were retrieved by pressure cooker and permeabilized by 20% tween for 20 min for fluorescence immunostaining. The nonspecific area was blocked with 0.1% BSA in 0.1% Triton X-100/PBS for 20 min. The sections were incubated overnight at 4^◦ C with Anti-MBP antibody (1:500, Abcam [ab218011], USA) and Anti-GFAP antibody (1:800, Abcam [ab68428], USA) and Anti-IDO-1 Antibody (1:250, EMD Millipore [MABF850], Germany), as primary antibody, in 0.03% PBS-Triton- × 100 supplemented with 5% normal goat serum (Abcam[ab7481-], USA). The slides were incubated with appropriate secondary antibodies (Alexa Fluor 488 and 566 (1:500, Thermo Fisher Scientific, USA), and Hoechst (1:1000, Abcam [ab228550], USA) for 1 h. Samples were analysed using a fluorescent microscope (Olympus IX-71; Olympus, Tokyo, Japan) equipped with a Canon EOS digital camera. For quantification of plaque number, we used soteriological dissector method for particle number. after preparation of immunohistochemically imaging of spinal cord, two adjust section with mentioned distance compared them (position and plaque size). The plaque with different position were counted. Totally number of each spinal cord plaque calculated for comparison between groups. Area of plaque estimated image J software.

After routine slicing of brain tissue, baked slices were dewaxed with xylene and then hydrated with a gradient ethanol solution, followed by heating for 30 min in citrate buffer (pH 6.0) to repair the high-temperature antigen. Sections were treated with 3% hydrogen peroxide for 20 min and blocked with goat serum for 30 min, followed by incubation at 4 °C overnight with rabbit anti-myelin basic protein (MBP; monoclonal; 1:5000; ab218011; Abcam, Cambridge, UK), rabbit anti-allograft inflammatory factor 1 (AIF1)/ionized calcium-binding adaptor molecule 1 (Iba-1; polyclonal; 1:100; Cat.# DF6442; Affinity, Jiangsu, China), and rabbit anti-A2aR (polyclonal; 1:200; 51092-1-AP; Proteintech, Rosemont, IL). After rewarming and incubation with the appropriate streptavidin–horseradish peroxidase (HRP)-conjugated secondary antibody for 20 min at 37 °C, the samples were stained 3,3′-diaminobenzidine, re-dyed, dehydrated, transparentized, and sealed. Images were visualized and obtained using a light microscope (Olympus) and analyzed using the ImageJ software (NIH, Bethesda, MD). A global threshold was manually established for the signal, followed by quantification of the positive signal detected above the selected threshold for the determination of an average optical density value.