Fluoroshield medium containing dapi

SH-SY5Y cells were seeded at 7 × 10⁵ cells/mL and differentiated on poly-D-lysine/laminin–treated coverslips, which were placed in a 12-well plate. Cells were treated with or without ghrelin, teaghrelin, and MPP⁺ for 24 h. The next day, cells were washed with phosphate-buffered saline (PBS) three times and immediately fixed with 4% paraformaldehyde for 1 h at room temperature. After cells were permeabilized for 20 min in 0.3% Triton-100, they were blocked with 1% BSA for 1 h and incubated with primary antibody against TH at 4 °C overnight. The next day, cells were washed three times with PBS and then incubated at room temperature for 1 h with anti-rabbit IgG Alexa 488 secondary antibody. Coverslips were mounted with fluoroshield medium containing DAPI (Abcam, Cambridge, MA, USA), and cells were evaluated using a fluorescence microscope (IX71; Olympus, FL, USA).

For immunofluorescence staining of spinal cord tissue, the spinal cord tissue was dehydrated in 30 % sucrose solution for 3 days, embedded in OCT, and cut into 10 μm thick sections in transverse section. Cryofixed spinal cord sections were permeabilized and blocked in TBST with 1 % BSA at ambient temperature for 1 h. The sections were then incubated overnight at 4 °C with the following antibodies: Anti-NeuN (1:300, Abcam, USA), Anti-CD68 (1:200, Abcam, USA), Anti-iNOS (1:200, Abcam, USA), and Anti-Arg1 (1:200, Abcam, USA). To visualize the sections, Cy3-labeled goat anti-mouse (1:500, Beyotime, China) and Alexa Fluor 488 goat anti-rabbit (1:500, Beyotime, China) were used as secondary antibodies. Nuclear detection was performed by applying Fluoroshield Medium containing DAPI (Abcam, USA). Finally, confocal microscopy was used to capture images of the sections.