Ab137869

Twelve-week-old C57 male mice were selected for model establishment. Mice were anesthetized with a 2,2,2-tribromoethanol solution before injection with 6-hydroxydopamine (3.3 μg/μl) using a stereotaxic apparatus. Injections were performed at the following two coordinates: anteroposterior (AP), 0.3 mm; mediolateral (ML), −2.2 mm; dorsoventral (DV), −3 mm; AP, 1.1 mm; ML, −1.7 mm; DV, −2.9 mm. The 6-hydroxydopamine solution (2 μl) was injected at each point and the needle was left in place for 5 min to promote drug absorption and prevent reflux. Then, 20,000 IU penicillin was injected for the first 3 days after the operation to prevent surgical infection. One week after surgery, mice received an i.p. injection of apomorphine (0.5 mg/kg). After 5 min of acclimatization, rotational data were continuously recorded for 30 min and the number of revolutions of more than seven circles per minute was considered to be successful model establishment (Pan et al., 2015 (link); Niu et al., 2018 (link); Chen et al., 2020 (link)). Additionally, western blotting (Tyrosine Hydroxylase, Abcam, ab75875) (Henriques et al., 2020 (link)), HE staining, immunohistochemistry (Tyrosine Hydroxylase, Abcam, ab137869) (Sun et al., 2021b (link)), and Nissl staining were used to verify the reliability of the model.

The SH-SY5Y cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and blocked with serum to prepare for immunofluorescence staining. Primary antibodies TH (ab137869, Abcam) and α-syn (ab138501, Abcam) were applied, followed by an overnight incubation at 4 °C. After washing off excess antibodies, a fluorescent secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor 594) (ab150080, Abcam) was added. Post-wash, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were then imaged using a fluorescence microscope (Ts2-FC, Nikon, Japan).

Protein extraction was done with RIPA lysis buffer (150 mM NaCl, 1% IGEPAL CA-630, 0,5% sodium deoxycholate, 0,1% SDS, 50 mM Tris, pH 8.0) containing 1 × protease/phosphatase inhibitors. Samples were lysed in the TissueLyser (Qiagen) until the tissue was fully homogenised. Samples were incubated on ice for 30 min, followed by a centrifugation step (30 min, 4 °C, 18,000 g). The supernatant was collected, and centrifuged again (20 min, 4 °C, 18,000 g). Then, supernatant was used to quantify the protein concentration with a Bradford assay (B6916, Sigma). The following primary antibodies were used: anti-tyrosine hydroxylase (ab137869, Abcam, diluted 1:2000), anti-glycerol kinase (ab126599, Abcam, diluted 1:2000) and anti-alpha tubulin (sc-23948, Santa Cruz, diluted 1:2000). The following secondary antibodies were used: goat anti-rabbit IgG, HRP (ab6721, Abcam, diluted 1:10,000), goat anti-mouse IgG, HRP (AP130P, Sigma, diluted 1:10,000), mouse IgG AF790 (ab175782, Abcam, diluted 1:10,000) and rabbit IgG AF680 (ab175772, Abcam, diluted 1:10,000). Immunoblots were visualised with chemiluminescence for HRP-linked secondary antibodies (Clarity™ Western ECL Substrate, 1,705,060, BioRad) or with fluorescence for the Alexa Fluor labelled secondary antibodies.

Following the collection of tissue, the samples were immersed in 4% paraformaldehyde for 48 h. Paraffin wax was used to embed them, which was then cut into sections of 5 μm. These sections underwent a gradient dewaxing and antigen retrieval using a citric acid solution. The activity of endogenous peroxidase was inhibited by applying 3% hydrogen peroxide for 15 min. Following rinsing, the sections were obstructed using a 3% BSA solution for 30 min. Subsequently, they were incubated with the primary antibody at 4 °C overnight. The next day, paraffin sections were brought to room temperature for 30 min and rinsed with PBS three times, each lasting 5 min. Afterwards, the second antibody was added gradually and left to incubate at ambient temperature for 50 min. After washing, DAB was added dropwise, and the reaction was terminated by microscopic control of color development time and rinsing after the appearance of positivity. Hematoxylin staining was rinsed after 1 min, and the hematoxylin differentiation solution was applied for several seconds. After dehydration and sealing, microscopic observation was performed, and data were collected. The following antibodies were used: PGP9.5(1:200, Cat# ab108986, Abcam), TH (1:200, Cat# sc25269, Santa Cruz), CHAT (1:200, Cat# ab137869, Abcam).