Sp5 upright confocal

Fluorescent and bright field images were taken using a Leica DM5000B with a Leica DFC500 camera. For confocal images, a Leica SP5 Upright Confocal was used.

Cells were seeded and grown onto Lab-Tek II chamber slides for 24 hr before the experiment. After washing two times with cold PBS, cells were fixed by treating at RT for 20 min with 2% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS containing 10% FBS for 10 min and blocked with 2% BSA for 1 hr. After washing four times with PBS, primary antibodies were added into the chambers, and incubated overnight at 4°C. The cells were washed again with PBS, followed by incubation with the secondary antibody for 1 hr at RT. The cells were finally washed again with PBS for four times and mounted with ProLong Diamond Anti-fade Mountant with DAPI (Molecular Probes). Slides were cured for 2 hr at RT and stored at 4°C overnight before imaging under microscope (× 63 oil lens, Leica Upright Confocal SP5). Protein co-association was quantified using Coloc2 plugin in Fiji software to determine the mean Pearson’s Correlation Coefficient R value (PCC R value). Background was subtracted by using a Rolling-Ball Background Substraction, threshold regression was set as costes and the costes randomizations were set at 10. The following primary antibodies were used for the immunofluorescence: HER2 (Zymed; 28004; 1:50), HER2-FITC (BD; 340553; 1:200), GRP94 (Sigma; G4420; 1:100), EEA1 (Abcam; ab70521; 1:100) and Calnexin (Abcam; 22595; 1:100).