Immunofluorescence Microscopy Protocol

The immunofluorescence technique was done as previously described [26 (link)]. Shortly, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) and permeabilized in cold methanol (− 20 °C). Then, cells were incubated with blocking buffer (5% Horse Serum in PBS) for 1 h. Cells were incubated with primary antibodies overnight and with secondary antibodies for at least 1 h and washed twice with PBS in between incubations. Hoechst 33258 (Sigma-Aldrich) or DAPI (Sigma-Aldrich) was used together with the secondary antibodies in order to stain the nuclei. Cover slips with cells were mounted in polyvinyl mounting medium. Cells were imaged using an Eclipse E800 Nikon or AxioImager Carl Zeiss microscopes and photographed with either a SPOT CCD or a Pursuit CCD camera (both from Diagnostic Instruments) using the manufacturer’s software. The images were analyzed and merged using Adobe Photoshop CC.