Donkey serum

For immunohistochemistry, animals were deeply anesthetized using 2% avertin and perfused with 0.1 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde at 4°C for 24 h and 30% sucrose 4°C for 48 h. Brains were then cut in coronal sections of 30 μm on a cryosection. The sections were blocked in 0.1 M PBS containing 0.3% Triton X-100 (Sigma) and 2% donkey serum (Genetex) for 30 min at room temperature. Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology). The brain samples with primary antibodies were incubated overnight at 4°C. Then, the sections were washed three times in 0.1 M PBS and incubated in proper secondary antibodies from the Jackson Laboratory for 1.5 h. After three rinses in 0.1 M PBS and DAPI staining at 1:3,000 (PIERCE), the sections were mounted on polysine microscopic slide glass (Thermo Scientific).
For immunocytochemistry, we fixed the cultured astrocytes with 4% paraformaldehyde at 4°C for 10 min. After washing with 0.1 M PBS three times, we performed immunocytochemistry according to the same procedures as immunohistochemistry.

Adult mice were deeply anesthetized with 2% avertin and perfused with 0.1 M PBS followed by 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde at 4°C for 24 hr and 30% sucrose at 4°C for 48 hr. Brains were then cut into 30 μm coronal cryosections. Sections were blocked in 0.1 M PBS containing 0.3% Triton X-100 (Sigma) and 2% Donkey Serum (GeneTex) for 30 min at room temperature. Primary antibody was then applied at the appropriate dilution and incubated overnight at 4°C. Sections were then washed three times in 0.1 M PBS and incubated in secondary antibody for 2 h. After three rinses in 0.1 M PBS and DAPI staining at 1:1000 (Pierce), the sections were mounted on polysine microscopic glass slides (Thermo Scientific). Images were acquired using a Nikon A1R confocal microscope.

Saline- and MPTP-injected mice were perfused with perfusion solution and postfixated with 4% paraformaldehyde for 1 day. The perfusion solution was made with NaCl, NaNO₃, and heparin (Sigma-Aldrich) in dissolving distilled water. The brains stored in 30% sucrose for cryoprotection were cut to a thickness of 30 μm. Dissection of the brain to obtain the SNpc and striatum regions was performed as previously described [17 (link)]. Brain slices were fixed with tissue stock solution and rinsed three times in phosphate-buffered saline (PBS pH 7.4). 0.3% Triton X-100 and 2% donkey serum (GeneTex, Irvine, CA, USA) in PBS were used for blocking for 90 min, then brain slices were incubated with primary antibodies against anti-tyrosine hydroxylase (Millipore, AB152) at 4°C overnight. For immunohistochemistry, brain slices were incubated with rabbit secondary antibodies (Dako EnVision⁺ System-HRP, USA) for 90 min, and then, reacting with DAB⁺ substrate buffer. After mounting by fluorescent mounting medium (Dako North America Inc., Santa Barbara, CA, USA) on cover slides, immunofluorescent images were acquired using an Olympus™ microscope (Olympus, Hachioji-shi, Tokyo, Japan).