HCT116 cells were grown in 8-wells culture slides and were fixed with 4% paraformaldehyde for 30 min. Slides, after permeabilization with triton X-100 0.1%/PBS for 10 min, were blocked with 3% normal goat serum/PBS for 30 min at room temperature. They were then incubated with anti-CD147 antibody (mouse monoclonal anti-human, dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C, followed by incubation with mouse secondary antibody (dilution 1:500, Alex Fluor 633 conjugated, clone 1H6, Thermo Fisher Scientific UK Ltd.) for 30 min. Afterwards, slides were incubated with fluorescein isothiocyanate–labeled phalloidin for 45 min to counterstained cytoskeleton. Subsequently, the slides were mounted with DAPI (Fluoromount G with DAPI; Electron Microscopy Sciences, Hatfield, PA, USA). Control samples were incubated with secondary antibody only. Slides were examined using an inverted confocal microscope (Nikon A1 MP, Instruments Inc. Melville, NY, USA). Images were taken using ImageJ software version 1.41 (NIH, Bethesda, Rockville, MD, USA). Analyses were performed using the OrientationJ plugins to analyze the coherency [19 (link)]. Quantification of nuclear and cytoplasm fluorescence signal was performed using the “Intensity Ratio Nuclei Cytoplasm Tool” plugin of Image J (NIH, Bethesda, MA, USA).
Free full text: Click here
