Motorized inverted research microscope model ix81

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.

The 3D basement membrane culture assay was performed according to the method previously described by Lee et al.^{17 (link)}, with slight modifications. Wells were pre-coated with 25 µl of Cultrex BME (Trevigen; 3432-005-01); once the coating has gelled, cells were resuspended in Cultrex BME and transferred to the coated wells (55 µl/well). Medium supplemented with 10% FBS and containing the indicated treatment was added on top of the embedded cells. Cells were then allowed to grow for 8–9 days; treatments were refreshed every 3–4 days. Images were obtained using an Olympus motorized inverted research microscope Model IX81 (×4 magnification), and quantified using the 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.