Mouse monoclonal anti th

Rats processed for immunohistochemistry were perfused with 0.9% saline and then with cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was removed and then washed and cryoprotected in the same buffer containing 20% sucrose. Brains were then cut into 40 μm sections on a freezing microtome. The sections were incubated for 1 h in 10% normal swine serum with 0.25% Triton X-100 in 20 mM potassium phosphate-buffered saline, containing 1% bovine serum albumin (KPBS-BSA), and subsequently incubated overnight (at 4 °C) with anti-tyrosine hydroxylase (TH) as the dopaminergic marker (mouse monoclonal anti-TH, Sigma-Aldrich Cat# T2928, RRID: AB_477569; 1:10,000). Sections were then incubated with the corresponding biotinylated secondary antibody (horse anti-mouse, Vector Laboratories, Inc., Newark, CA, USA; Cat# BA-2001, RRID: AB_2336180; 1:200) for 60 min, and then with an avidin–biotin–peroxidase complex (ABC, 1:100, Vector) for 90 min. Finally, sections were revealed with 0.04% hydrogen peroxide and 0.05% 3-3′diaminobenzidine (DAB, D5637, Sigma-Aldrich). All experiment control sections in which the primary antibody was omitted were immune-negative for TH.

Following the behavioral tests, the animals were deeply anesthetized and perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde (PFA). Mouse brains were immediately removed from the skull and post-fixed in PFA solution overnight. After that, brains were dehydrated in a serial of alcohol solution and dimethylbenzene was applied to make the brains transparent. Brains were then embedded in paraffin wax. Next, 4 μm thickness sections were cut through SN compacta (SNpc), and every fourth slice was reserved. For staining, sections were deparaffinized and rehydrated, followed by epitope retrieval by boiling the sections in 0.01 M citrate buffer (pH 6.0) for 10–15 min and cooling at room temperature for 20–30 min. After that, sections were thoroughly washed in 0.01 M PBS three times. Endogenous peroxidase activity was blocked with 3% H₂O₂ in 0.01 M PBS for 30 min. Next, the sections were incubated in 5% BSA with 0.1% Triton X-100 in PBS for 30 min, followed by incubation with mouse monoclonal anti-TH (1:1000, Sigma, St. Louis, MO, United States) at 4°C overnight. Subsequently, the sections were washed in PBS, incubated with goat anti-mouse secondary antibody at room temperature for 1 h and stained using a DAB kit (Gene Tech, GK500705, China). The sections were observed and photographed using the Zeiss microscope (Carl Zeiss, 37081, Germany).