For immunofluorescence (IF), HeLa cells were grown on 12-mm coverslips at an initial density of 2.5 × 105 per well on 6-well plates. At 48 h after transfection, cells were fixed with 4% PFA in phosphate-buffered saline (PBS) for 15 min at room temperature (RT), permeabilized with 0.5% Triton X-100 in PBS (v/v) for 5 min at 4 °C, and blocked with 1% FBS in PBS (for 30 min at RT). All wash steps were done with PBS.
Primary antibodies were used for overnight incubation at 4 °C and secondary antibodies for 60 min at RT. DNA was stained with DAPI (4,6-diamidino-2-phenylindole, 0.2 μg/mL in mounting medium with Mowiol and DABCO). For imaging, the confocal microscopes LSM 510 META with the FCS system and Olympus FluoView FV1000 were used. Any brightness and contrast adjustments were performed in Adobe Photoshop, Zen 2007 (Carl Zeiss, Oberkochen, Germany) or ImageJ [64 (link)].
The fibroblasts were seeded on 12-mm coverslips to obtain 50% confluence, then fixed after 24 h and stained as described for HeLa cells. For imaging, the confocal microscope Leica SP8 (Leica Camera AG, Wetzlar, Germany) with LasX software was used. Z-stacks were presented as maximum intensity projection images.
Primary antibodies were used for overnight incubation at 4 °C and secondary antibodies for 60 min at RT. DNA was stained with DAPI (4,6-diamidino-2-phenylindole, 0.2 μg/mL in mounting medium with Mowiol and DABCO). For imaging, the confocal microscopes LSM 510 META with the FCS system and Olympus FluoView FV1000 were used. Any brightness and contrast adjustments were performed in Adobe Photoshop, Zen 2007 (Carl Zeiss, Oberkochen, Germany) or ImageJ [64 (link)].
The fibroblasts were seeded on 12-mm coverslips to obtain 50% confluence, then fixed after 24 h and stained as described for HeLa cells. For imaging, the confocal microscope Leica SP8 (Leica Camera AG, Wetzlar, Germany) with LasX software was used. Z-stacks were presented as maximum intensity projection images.