HeLa cells were grown on coverslips prior to processing for IF. Cells were fixed onto coverslips in 4% paraformaldehyde (Thermo Fisher Scientific [Waltham, MA, USA]) for 20 min at room temperature, then permeabilized in 0.1% Triton X-100 (MilliporeSigma [Burlington, MA, USA]) for 10 min. After blocking in 4% bovine serum albumin for 15 min, cells were incubated for 45 min with primary antibodies diluted 1:500 in blocking solution. After exhaustive washing in phosphate buffered saline (PBS), cells were incubated in fluorescent secondary antibodies diluted 1:700 in blocking solution. Prior to mounting onto slides, the cells were stained with Hoechst 33342 (Life Technologies [Carlsbad, CA, USA]) and adhered to slides with Fluoromount-G (Southern Biotech [Birmingham, AL, USA]).
Confocal images were collected on an LSM 880 laser scanning microscope (Zeiss [Jena, Germany]) with a C Plan-Apochromat 63×/1.40 oil DIC M27 objective using ZEN 2.1 software (Zeiss [Jena, Germany]). Image quantification and processing was performed in Fiji [67 (link)] or CellProfiler [68 (link)] as described below.
Confocal images were collected on an LSM 880 laser scanning microscope (Zeiss [Jena, Germany]) with a C Plan-Apochromat 63×/1.40 oil DIC M27 objective using ZEN 2.1 software (Zeiss [Jena, Germany]). Image quantification and processing was performed in Fiji [67 (link)] or CellProfiler [68 (link)] as described below.