The cells were fixed with methanol at −30°C for 20 min or with 2% paraformaldehyde for 20 min, followed by permeabilization with 0.2% Triton X-100. After incubation with blocking buffer (0.2% Tween-20, 1 mg/ml bovine serum albumin, PBS) for 60 min at room temperature, the cells were incubated overnight at 4ºC with the indicated primary antibodies and for 1 h at room temperature with the indicated secondary antibodies. The cells were observed using a 1.4 numerical aperture CFI Plan-Apo VC 60× or Plan-Apo 63× oil immersion objective lens under a confocal laser microscope (LSM780; Carl Zeiss, Jena, Germany).
To quantify the disruption of the Golgi ribbon, the circularity index of the Golgi morphology was measured as described elsewhere (Miller et al., 2009 (link)). Briefly, a freehand selection option in ImageJ software (National Institutes of Health, Bethesda, MD) was used to outline the Golgi based on GM130 staining. Circularity index values were assigned to Golgi outlines by the ImageJ circularity plug-in (http://rsb.info.nih.gov/ij/plugins/circularity.html), where circularity = 4π(area/perimeter2). A circularity value of 1 corresponds to a perfect circle.