Ab75875

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.

Substantia nigra Sect. (5 μm) from each group were dewaxed and dehydrated. Then, the sections were treated with 3% BSA for 1 h at 25 °C and incubated with rabbit anti-mouse tyrosine hydroxylase (TH) (1:100, ab75875, Abcam, UK) overnight at 4 °C. After washing the sections with PBS, they were incubated with goat anti-rabbit IgG antibody (1:500, s0001, Affinity, AUS) at room temperature for 2 h, stained with diaminobenzidine (DAB, Solarbio, China), and counter-stained with hematoxylin. The TH-positive area of the substantia nigra was observed under a microscope, and five fields were randomly selected for image analysis. Immunohistochemical images were quantified using Image J (NIH, USA) analysis software. Mean density, which represents the expression of protein, is the cumulative integrated optical density (IOD) divided by the area of the effective target distribution.

We performed immunofluorescence experiments on horizontal cryo‐sections of 25 μm thickness and proceeded as previously described (Tozzini et al., 2012 (link)). Briefly, we washed the sections in PBS to remove the cryo‐embedding medium; then, we performed an acid antigen retrieval step (Tri‐sodium citrate dehydrate 10 mM, tween 0,05%, pH 6). Afterward, we stained the sections with Aggresome (ProteoStat™ Aggresome Detection Kit, Enzo Life Sciences Inc.; for more details see Shen et al., 2011 (link)) as follows: We applied a solution 1:2000 of Aggresome dye in PBS for 3 min, rinsed in PBS and left the sections immersed in 1% acetic acid 40 min for de‐staining. We applied blocking solution (5%BSA, 0,3% Triton‐X in PBS) for 2 h, then the primary antibody at proper dilution in a solution of 1% BSA, 0,1% triton in PBS, and incubated the samples overnight at 4°. After rinsing in PBS, the following day secondary antibody was applied at a 1:400 dilution in the same solution used for the primary antibody. After 2 h, slides were rinsed 3 times with PBS and mounted with a specific mounting medium added with nuclear staining (Fluoroshield DAPI mounting, Sigma‐Aldrich). The antibodies utilized were thyroxine hydroxylase (Rabbit Monoclonal, ab75875, Abcam), Alexa Fluor 568 Goat anti‐Mouse (A11004), and Alexa Fluor 488 Goat anti Rabbit (A11008) (Invitrogen).