Nunc lab tek glass chamber slide system

For PLA cells were plated in 16-well Nunc Lab-Tek glass Chamber Slide System (178599; Thermo Scientific) and treated as indicated for 2 days. Following fixation, cells were incubated with rabbit anti-ErbB2 and mouse anti-nucleolin antibodies. PLA was performed using the Duolink In-Situ PLA probes: anti-rabbit MINUS and anti-mouse PLUS, and the Duolink In-Situ Detection Reagents Red kit (DUO92005; DUO92001; DUO92008, respectively; Sigma-Aldrich), according to the manufacturer’s instructions. Nuclei were stained using the Duolink In-Situ Mounting Medium with DAPI (DUO82040; Sigma-Aldrich). Slides were visualized 24 h post staining and images were obtained using an Olympus motorized inverted research microscope Model IX81 (×60 magnification). Signal intensity was determined using ImageJ software.

For proximity ligation assay (PLA) cells were plated in 16-well Nunc Lab-Tek glass Chamber Slide System (178599; Thermo Scientific) and grown for 48-72h with or without siRNA treatment, as indicated. Following fixation, cells were incubated with rabbit anti-ErbB2 and mouse anti-nucleolin antibodies. PLA was performed using the Duolink In-Situ PLA probes: anti-rabbit MINUS and anti-mouse PLUS and the Duolink In-Situ Detection Reagents Red kit (DUO92005; DUO92001; DUO92008, respectively; Sigma-Aldrich), according to the manufacturer's instructions. Nuclei were stained using the Duolink In-Situ Mounting Medium with DAPI (DUO82040; Sigma-Aldrich). Slides were visualized 24h post-staining and images were obtained using an Olympus motorized inverted research microscope Model IX81 (60× magnification). Signal intensity was determined using ImageJ software.