Immunofluorescence Labeling of Cellular Organelles

For all images shown, cells were fixed for 20 min in 4% formaldehyde/PBS and permeabilized in 0.5% Triton X-100/PBS. For Table 2, cells were also fixed in methanol (5 min) and acetone (45 s), or as above but with 0.1% glutaraldehyde/2% paraformaldehyde/PBS and after permeabilization quenched with 1 mg/ml NaBH₄ in PBS for 5 min. Cells were blocked for 30-60 min in PBS containing 20% FCS and 0.25% Tween-20 and probed with the antibodies in the same buffer. Rabbit antibodies against Arl8 (Hofmann and Munro, 2006 (link)), GM130 (Abcam, ab30637, 1:500 dilution), Golgin245 (Sinka et al., 2008 (link)), Sec16 (Ivan et al., 2008 (link)), and the mouse and goat (1:2000) antibodies generated in this study were detected with species-specific Alexa-labeled secondary antibodies (Molecular Probes, 1:400), or IgG subclass-specific secondary antibodies (Invitrogen). For standard imaging, cells were mounted in VECTASHIELD (Vector Laboratories) and micrographs obtained with a confocal microscope (Zeiss LSM 780) or a wide-field microscope with Micro-Manager software [Zeiss Axioplan and CoolSNAP HQ2 (Photometrics)]. For STED microscopy, coverslips were mounted in Prolong Gold anti-fade mounting media (Invitrogen). Super resolution STED images were acquired with a LEICA TCS SP8 X microscope equipped with 592 nm and 660 nm depletion lines.