2.9. Fluorescence Microscopy
For immunofluorescence (IF), cells grown on coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature and permeabilized with 0.2% Triton X-100 for 10 min. Cells were blocked in phosphate-buffered saline (PBS) containing 1% BSA, incubated with the indicated primary antibodies for 1 h at 37 °C, rinsed with PBS and then incubated with Alexa Fluor-tagged secondary antibodies for 1 h at 37 °C. Cell nuclei (DNA) were stained with Hoechst 33,342 (Sigma-Aldrich, St. Louis, MO, USA). Images were acquired separately for each channel using a Zeiss Axiovert 200 M microscope equipped with a Plan-Apochromat 100× (1.4 NA) and a Zeiss Axiocam digital camera (Zeiss, Oberkochen, Germany). AxioVision software 4.7.1 (Zeiss, Oberkochen, Germany) was used for merging pictures, and Tiffs were assembled into figures using Adobe Photoshop CS6. For quantitative colocalization analysis (QCA), digital photographs were quantitated using the AxioVision colocalization software (4.7.1; Zeiss, Oberkochen, Germany) by calculating the pixel intensity-based Pearson’s correlation coefficients (PCC) of randomly selected cells (25 cells per coverslip).