Dapi fluoromount g

For immunofluorescence, cells were grown on glass coverslips in 12-well plate. After treatment with indicated concentration paeoniflorin for 24h, the cells were fixed with 4% formaldehyde for 15 minutes at room temperature and washed in PBS for 3×5 minutes. Then 1× Phalloidin-iFluor 488 was added and incubated 90 minutes at RT. After being washed in PBS for 3×5 minutes, coverslips were mounted on glass slides using mounting medium (DAPI Fluoromount-G, Thermo Fisher Scientific, USA). At last, the images were obtained with the Laser Scanning Confocal Microscope (Leica, Germany).

Cells were grown to 40% confluence in 8-well glass chamber slides (Nunc, Thermo Fisher Scientific), prior to growth arrest and treatment. Cells were washed with PBS before fixation in 4% paraformaldehyde for 10 min at room temperature. Next, cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature. For visualization of ECM, cells were fixed with 100% ice-cold methanol for 8 min at −20 °C. Slides were washed with PBS and blocked with 1% goat serum (TBD Science, Tianjin, China) for 30 min before washing with 0.1% (w/v) BSA in PBS. Subsequently, slides were incubated with monoclonal mouse anti-α-SMA antibody (Sigma-Aldrich, 1:50) diluted in 0.1% BSA-PBS, overnight at 4 °C. After further wash steps, slides were incubated with goat anti-mouse IgG CF-488 secondary antibody (Abcam, 1:1000) in 0.1% BSA-PBS for 1 h at room temperature and under darkness. For F-actin visualization, after fixation and permeabilization; cells were incubated with phalloidin conjugated to FITC (Sigma-Aldrich) in 0.1% BSA-PBS, overnight at 4 °C. Cells were then mounted in DAPI Fluoromount-G (Thermo Fisher Scientific) and imaged using a Zeiss Axio Imager Z1 UV fluorescence microscope (Zeiss, Jena, Germany) and a Leica TCS SP5 laser scanning confocal microscope (Leica Biosystems, Baden-Württemberg, Germany).