Edu alexa fluor kit

To determine the effect of patulin on LC proliferation, EdU incorporation into R2C cells was utilized with the EdU Alexa Fluor Kit (Life Technologies, Carlsbad, CA, USA). The R2C cells were treated with 0–4 μM patulin for 24 h as detailed above. EdU (diluted 1:1000, v/v) was incubated for an additional 24 h, followed by fixation in 4% paraformaldehyde for 30 min and reaction for the display labeled nucleus (green). LCs were visualized via the immunofluorescence of CYP11A1 (red) and 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA). Image acquisition was performed using a Leica automatic stage DM5500B microscope (Leica, Wetzlar, Germany), and the EdU-positive cells were counted using Image Pro Plus 7.0 software.

EdU incorporation into stem Leydig cells on the surface of the seminiferous tubules was measured using EdU Alexa Fluor Kit (Life Technologies, USA) as previously described.23 In brief, the freshly isolated seminiferous tubules as above were cultured in M199 medium and treated with 0, 1, 10 ng/mL FGF16 for 7 days. Then, 1:1000 (v/v) diluted EdU was added to the well having seminiferous tubules and EdU incorporation lasted for 24 hours. Tubules were washed with PBS, fixed in 4% paraformaldehyde, incubated with the reaction solution, and mounted on a glass slide. The EdU staining was visualized under a fluorescence microscope (Olympus, Japan) and images were captured. EdU‐positive (green colour in the nucleus) cells on the surface of the tubules were counted using the ImageProPlus 7.0 software (Media Cybernetics, Rockville, MD, USA).