Tgn46

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).

For cell culture, primary and secondary antibodies were used at 1:300 concentrations. EGFR clone 225 (Milipore). MUC-1 Ab-5 (Thermo Fisher Scientific). EEA1 H-300 (Santa Cruz Biotechnology). COX-IV (Cell Signaling 3E11). TGN46 (Sigma 7576). FAK A-17 (Santa Cruz) was used at a 1:50 primary and 1:100 secondary concentration. For mammary glands, primary antibodies were used at 1:300 concentrations; secondary antibodies were used at 1:500 concentrations. EGFR 1005-G (Santa Cruz Biotechnology). AlexaFluor 488 donkey anti-mouse IgG (Invitrogen), AlexaFluor 594 donkey anti-rabbit IgG (Invitrogen), AlexaFluor 594 donkey anti-goat IgG (Invitrogen), AlexaFluor 647 goat anti-armenian hamster IgG (Jackson ImmunoResearch).

Tissue specimens were washed in cold PBS, fixed in 10% formalin buffered saline for 2 hr, followed by incubation in 30% sucrose overnight. Tissue specimens were embedded in optimal tissue cutting medium and 6-µm-thick section were cut. Sections were rinsed in PBS followed by antigen retrieval in 0.05% citraconic anhydride (Sigma-Aldrich, St Louis, MO), permeabilized in 0.05% Triton X-100 or 0.05% saponin (TGN46) (Sigma-Aldrich, St Louis, MO), washed in PBS, and blocked in 5% goat serum (Sigma-Aldrich, St Louis, MO). Sections were incubated with primary antibodies toward EEA1, Rab7 (Cell Signaling Technology, Beverly, MA), Lysozyme, TGN46 (Thermo Fisher Scientific, Waltham, MA), Rab3D (Synaptic Systems, Goettingen, Germany), LAMP1, Calnexin (Abcam, Cambridge, United Kingdom), or VAMP-8 (gift from Prof Burton Dickey, University of Texas) at 4°C overnight, rinsed with PBS and incubated with goat anti rabbit or goat anti chicken Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA) at room temperature for 1 hr. Sections were counterstained with DAPI and imaged using an Axioskop 2 microscope, an LSM700 Axio Examiner Z1 confocal microscope, or an LSM900 with Airyscan 2 microscope (Carl Zeiss Microscopy, Thornwood, NY). Acquired images were analyzed using Zen (Carl Zeiss Microscopy, Thornwood, NY) and Imaris (Bitplane, Belfast, Great Britain) software.