Zli 9062

Both PC-3 and LNCaP cell lines were subcultured until they reached 70% confluency; they were then washed twice with phosphate-buffered saline (PBS) (ZLI-9062; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and dissociated with 0.25% Trypsin (ZLI-9010; Zhongshan Golden Bridge Biotechnology Co. Ltd.). Before being treated with zinc (ZnCl₂; 012307; Sigma-Aldrich, St. Louis, MO, USA), both cell lines were seeded in 24-well plates (FCP243; Beyotime Biotechnology Co., Ltd., Shanghai, China) at a density of 1×10⁵ cells per well and allowed to attach overnight. Cells were subsequently treated with zinc (100 µM) for 0, 4, 8, 12, or 24 h.

Immunohistochemistry was carried out as previously described [23 (link)]. Mice were anesthetized with 8% chloral hydrate and perfused with physiological saline followed by 4% paraformaldehyde. Then, the brain was fixed in 4% paraformaldehyde and embedded in 30% sucrose, then cut into 20-µm thick coronal sections. Sections were rinsed three times in phosphate-buffered saline (PBS; Zhongshan Golden Bridge Biotechnology Co., PV-9002, ZLI-9062) and then incubated in 3% H2O2 for 15 min to block endogenous peroxidase activity. After washing three times in PBS, sections were incubated in 10% goat serum (Cell Signaling Technology, 5425) followed by permeabilization with 0.3% TritonX-100 (Sigma-Aldrich, T9284) for 60 min at room temperature and incubation overnight at 4°C in anti-TH antibody. A biotinylated goat anti-mouse secondary antibody (1:200; Zhongshan Golden Bridge Biotechnology Co., PV-9002) and diaminobenzidine (Zhongshan Golden Bridge Biotechnology Co., ZLI-9019) were used to detect immunoreactivity. We selected 20 successive sections from each brain for examination. The total number of TH-positive neurons was determined using the optical fractionator probe. Three mice from each group were used for this measurement.