Tcs sp8 sted confocal laser scanning microscope

For immunofluorescence analysis, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization with 0.3% Triton X-100 in PBS for 5 min. The cells were blocked with 10% goat serum (ZSGB-BIO, Beijing, China) for 60 min at room temperature. The cells were then incubated with the primary antibodies at 4 °C overnight. Following three 5-min washes in PBS with gentle agitation, an Alexa Fluor-conjugated secondary antibody (Invitrogen) at 1:500 was added, and the samples were incubated for 1 h at 37 °C. The nuclei were counter-stained with DAPI (Sigma-Aldrich). The images of fluorescently labelled were captured using a Leica TCS SP8 STED confocal laser scanning microscope and a Leica TCS SP5 confocal laser scanning microscope (Leica, Wetzlar, Germany). Relative intensities of staining were quantitatively assessed using Image J software.
To visualize F-actin, the cells were stained with Alexa Fluor 568-conjugated phalloidin (Invitrogen) for 30 min at room temperature. The images of fluorescently labelled were captured using a Leica TCS SP8 STED confocal laser scanning microscope and a Leica TCS SP5 confocal laser scanning microscope (Leica, Wetzlar, Germany). The fluorescent co-localization between F-actin signal and vinculin was quantified by Pearson’s coefficient analysis using ImageJ ‘Colocalization Threshold’ software.

Cells were plated on confocal chambers (NEST) at a density of 35,000 cells per well. Next, cells were stained with Hoechst 33,342 (0.2 μg/ml, Thermo Fisher Scientific, Waltham, MA, USA) for 20 min, following incubation, 50 nM MitoTracker Green FM (M7514, Thermo Fisher Scientific) or 25 nM tetramethylrhodamine methyl ester (TMRM) (I34361, Thermo Fisher Scientific) was added, for 30 min at 37 °C, in a CO₂ incubator, then washed three times with PBS. After washing, the images of fluorescently labelled mitochondria and nuclei were captured using Leica TCS SP8 STED confocal laser scanning microscope (Leica). The relative intensities of Mito Tracker Green FM and TMRM were calculated in each cell using ImageJ software as previously described [50 (link)].