Immunofluorescence Analyses of Cellular Structures

Immunofluorescence analyses were carried out as previously described [12 (link),40 (link)]. Preparations were first incubated with the primary antibodies in PBS containing 0.1% bovine serum albumin (BSA) at room temperature for 1 h and, after extensive washing with PBS, they were incubated with the secondary antibodies also in PBS containing 1% BSA at room temperature for 45 min. Preparations were finally mounted in 4′,6-diamidino-2-phenylindole (DAPI)-containing ProLongTM Gold antifade (Invitrogen). Fluorescence signals were captured with either a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) or a DeltaVision Elite system (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
Primary antibodies used in this study were monoclonal antibodies against dsRNA (English and Scientific Consulting Kft, Szirák, Hungary), TGN46 (Sigma) and PDI (Enzo, Farmingdale, NY, USA), and polyclonal antisera against GIANTIN (Abcam, Cambridge, UK) and GM130 (Abcam). Secondary antibodies were Alexa488- or Alexa568-conjugated goat anti-rabbit or anti-mouse antibodies (Life Technologies, Waltham, MA, USA).