Tyrosine hydroxylase is a rate-limiting enzyme in the synthesis process of dopamine and norepinephrine (Moore and Bloom, 1979). It is abundantly expressed in dopaminergic neurons (Raisman-Vozari et al., 1991; Blanchard et al., 1993). The protocol for immunofluorescence staining of TH is routinely used in our laboratory (Jiang et al., 2008; Shen et al., 2017). Brains were fixed in 4% PFA for 72 h at 4°C then incubated in 0.1 mol/L phosphate buffer (pH 7.5) containing 25% sucrose at 4°C for 2–3 days. The frozen brain tissues were cut into 20-μm-thick sections. The brain tissue sections were used for immunofluorescence staining of TH in the SNpc. The free-floating sections were first incubated with 0.1% Triton X-100 and goat serum (Gibco-BRL, Grand Island, NY, United States) in phosphate-buffered saline (PBS) for 2 h and then incubated overnight at 4°C with the TH primary antibody (1:1,000) (Millipore, Burlington, MA, United States) in PBS containing 0.1% Triton X-100 (St. Louis, MO, United States). The sections used for staining TH in the SNpc were incubated with Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (Abcam, Cambridge, United Kingdom), and images were obtained by immunofluorescence microscopy (Observer A1, Zeiss, Germany) at magnifications of 100× and 400×.

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