4 6 diamidino 2 phenylindole dapi staining solution

For immunofluorescence assays, cells were cultured on coverslips, fixed with 4% paraformaldehyde at 25 °C for 15 min, and then permeabilized with 0.5% Triton X-100 in PBS for 5 min. After blocking with 5% bovine serum albumin, the cells were incubated with the indicated primary antibodies for 1 h at 25 °C. The cells were then washed with PBS twice and incubated with goat anti-rabbit or goat anti-mouse fluorescently -labeled IgG (1:1000, Abcam, UK) for 1 h. The cells were counterstained with 100 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) staining solution (Sigma-Aldrich, USA) for 2 min to visualize nuclear DNA. The coverslips were mounted onto glass slides with FluorSave™ Reagent (Millipore, USA) and visualized under an Olympus IX73 Microscope Imaging System (Olympus, Japan).

HK-2 cells were seeded in a 6-well plate, grown to 90% confluency, and received indicated treatments for 3 h. The original medium was aspirated, and then 4′,6-diamidino-2-phenylindole (DAPI) staining solution (1 μg/L; Sigma, USA) prepared in methanol (1 mL) was then added to each well, followed by incubation at 37°C in a 5% CO₂ atmosphere for 20 min. The cells were washed three times with phosphate-buffered saline (PBS; Gibco, USA) and visualized under a fluorescence microscope (Olympus, Japan) at ×400 magnification. The number of apoptotic cells was counted using the ImageJ software.