Immunofluorescence Staining of U-2 OS Cells

U-2 OS cells grown on glass coverslips were washed with PBS, fixed in 3% paraformaldehyde for 15 minutes (at 37°C) and permeabilized 30 min in PBS containing 10% FCS and 0.1% saponin or briefly extracted with an extraction buffer (0.5% TX-100, 10% glycerol in EMT [60 mM PIPES-NaOH pH6.9, 25 mM HEPES, 10 mM EGTA, 10 mM MgCl₂]) to obtain cytoskeletons and fixed in 3% paraformaldehyde in PBS for 15 minutes (at 37°C). Samples were then processed for immunofluorescence as in [9 (link)]. The primary antibodies (anti-BILBO1 1–110, 1:4000 dilution; anti-living colours rabbit polyclonal Clontech, 1:1,000 dilution; anti-HA tag mouse monoclonal IgG1 Biolegend, 1:1,000 dilution) were incubated for 1h in a dark moist chamber. After two PBS washes, cells were incubated for 1h with the secondary antibodies anti-rabbit IgG conjugated to Alexa fluor 594 (Molecular Probes, 1:400); anti-mouse IgG conjugated to FITC (Sigma, 1:400). The nuclei were stained with DAPI (0.20 μg.mL^-1 in PBS for 5 min), then washed twice in PBS and mounted overnight with Prolong (Molecular Probes P-36934). Images were acquired on a Zeiss Imager Z1 microscope with Zeiss 100x or 63x objectives (NA 1.4), using a Photometrics Coolsnap HQ2 camera and Metamorph software (Molecular Devices), and processed with ImageJ.