Sc 516141

Sperm were spread onto glass slides and fixed with methanol for 10 min. Then, the samples were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS). Nonspecific binding was blocked with PBS containing 10% BSA (w/v) (Life Technologies, Grand Island, NY, USA) for 30 min at 25°C. The sperm were incubated with primary anti-4HNE (ab48506; Abcam, Cambridge, UK) overnight. Next day, the sperm were washed three times in PBS and incubated with goat anti-mouse (1:100, sc-516141; Santa Cruz Biotechnology, Paso robles, CA, USA) antibody for immunofluorescence labeling. Subsequently, digital images were captured using a fluorescence microscope (80i; Nikon, Tokyo, Japan). The negative controls were treated without the primary antibodies.

After deparaffinization of the sections, antigen retrieval was accomplished by boiling in citric acid–sodium citrate buffer (0.01 M, pH 6.0) for 15 min, and endogenous peroxidase blocking was performed using the same method as described above. Then, sections were incubated with a working solution of primary rabbit anti-vimentin monoclonal antibody (ZA-0511; Zhongshan Golden Bridge Biotechnology Co., Beijing, China) at 4 °C for 12 h. Next, sections were incubated with an anti-rabbit secondary antibody (ZF-0511, diluted 1:400; Zhongshan Golden Bridge Biotechnology Co., Beijing, China) at 37 °C for 1 h. Then sections were incubated with primary mouse monoclonal antibody against CYP27B1 (sc-515903, diluted 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, US) at 4 °C for another 12 h. The anti-mouse secondary antibody (sc-516141, diluted 1:50; Santa Cruz Biotechnology, CA, US) was added and incubated at 37 °C for 1 h, then nuclei were counterstained with DAPI (Neobioscience Biological Technology Co., Shenzhen, China), and sections were observed using immunofluorescence microscopy (Nikon, Tokyo, Japan).

RAW 264.7 cells were plated in a chambered coverslip (18 wells) at a density of 2 × 10⁴ cells/well and separated into the following 4 groups: 1. No stimulation: cells were incubated in DMEM medium alone. 2. Stimulated: cells were cultured in DMEM medium + 20 ng/mL M-CSF and 20 ng/mL RANKL. 3. Experiment group: cells were incubated with Ps-Gos (0.00001–100 µg/mL) + 20 ng/mL M-CSF and 20 ng/mL RANKL. 4. Positive control: cells were treated with 30 µM D-pinitol + 20 ng/mL M-CSF and 20 ng/mL RANKL. After incubation for 24 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (No. 39487. Cell Signaling Technology, Danvers, MA, USA). After blocking with 5% goat serum (S26-100ML, Sigma Aldrich), they were incubated with the primary anti-mouse RANK antibody (1: 500; sc-390655, Santa Cruz Biotechnology) at 4 °C overnight with shaking. Subsequently, the cells were incubated with a secondary antibody (1:200; No. sc-516141, Santa Cruz Biotechnology) for 3 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (No. 4083, Cell Signaling Technology, Danvers, MA, USA) for 5 min. Finally, mounting medium was carefully added to preserve the fluorescence signal, and photographs were taken with a fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence intensity was analysed with ImageJ software.