Infected Huh-7, HeLa cells or primary mouse hepatocytes were fixed at the indicated time points either in ice-cold methanol/acetone (1:1) at −20°C for 5 min without further permeabilization step or in 4% paraformaldehyde (PFA) in phosphate-buffered saline [PBS, 137 mM NaCl (Sigma-Aldrich, S9888-1Kg), 2.7 mM KCl (Fluka Chemie AG P9541-1 kg), 10 mM Na2HPO4 (Sigma-Aldrich, S5136-500 g), 1.8 mM KH2PO4 (Sigma-Aldrich, P5655), pH 7.4] for 10 min at room temperature and subsequently permeabilized with 0.1% Triton X-100 in PBS (Fluka Chemie, T8787-250 ml), for another 10 min. After 1 h blocking with 10% FCS-PBS, cells were stained with the diluted primary antibodies for an additional hour. For fluorescent labeling, cells were subsequently incubated with secondary antibodies for 1 h and nuclei visualized with 1 µg/ml DAPI (Invitrogen, D-1306). All incubations were performed at room temperature. For microscopy, cells were mounted on microscope slides using Dako Fluorescent Mounting Medium (Dako, S3023).

The primary antibodies used were: mouse monoclonal anti-GFP (Roche, 11814460001), rabbit polyclonal anti-GFP (Acris, SP3005P), rabbit polyclonal anti-UIS4 (kindly provided by Photini Sinnis, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA), chicken polyclonal anti-Exp1 (Heussler lab, Bern, Switzerland), mouse monoclonal anti-GM130 (BD-Biosciences, 610822), mouse monoclonal anti-Golgin-97 (Invitrogen, #A21270), rabbit polyclonal TGN46 (abcam #ab16059), mouse monoclonal anti-α tubulin (Sigma, T9026), and rat monoclonal anti-HA (Roche, 11867423001). All primary antibodies were used at a dilution of 1:500 in 10% FCS in PBS. As secondary antibodies, species-specific antibodies conjugated to Alexa Fluor® 488 (Invitrogen Molecular Probes, A-11001; A-11008), Alexa Fluor® 594 (Invitrogen Molecular Probes, A-21209; A-11032) or Cy5 (Dianova, 111-175-144; 703-175-155) were used at a final concentration of 0.2 µg/ml.

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