Mouse irdy594

Neurons or cells were fixed with 4% (w/v) paraformaldehyde and 4% (w/v) sucrose in PBS for 15 min. To extract soluble proteins, we added 1% (v/v) TX-100 to the fix buffer. The cells were rinsed five times with PBS and then permeabilized and blocked with 3% (w/v) BSA/0.1% (v/v) TX-100 for 15 min. Then the cells were incubated with the primary antibody [anti-α-synuclein (1:500; BD Biosciences, Franklin Lakes, NJ, USA) or anti-p-α-synuclein (1:400; Abcam, Cambridge, UK)] for 2 h at room temperature. After rinsing five times with PBS, the cells were incubated with a secondary antibody, mouse IRDy594, rabbit IRDy488, or rabbit IRDy647 (1:500; LI-COR, Lincoln, NE, USA), for 1 h at room temperature. The fluorescence was visualized with confocal microscope (Leica Microsystems, Tokyo, Japan).

Mice were anesthetized and perfused with physiological saline followed by 4% paraformaldehyde/PBS, and the brains was removed, followed by fixation in 4% paraformaldehyde overnight and transfer to 30% sucrose for cryoprotection. Then the brains were sectioned at a thickness of 40 μm, and the sections were incubated in 0.3% TX-100/PBS for 60 min and then incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity. After washing in PBS, sections were incubated in 10% goat serum followed by 0.1% TX-100 in PBS for 60 min, then incubated in anti-tyrosine hydroxylase (TH) (1:8,000; Sigma, St. Louis, MO, USA), anti-p-α-synuclein (1:400; Abcam, Cambridge, UK), anti-MAP2 (1:400; Abcam, Cambridge, UK), anti-GFAP (1:400; Abcam, Cambridge, UK), or anti-Iba1 (1:200; Wako Pure Chemical Industries, Japan) antibodies for 2 h at room temperature. After washing in PBS, the brain slices were incubated with secondary antibody, mouse IRDy594 or rabbit IRDy488 (1:500; LI-COR, Lincoln, NE, USA), for 1 h at room temperature. The fluorescence was visualized and analyzed with confocal microscope (Leica Microsystems, Tokyo, Japan).