Alexa fluor 555 conjugated donkey anti mouse secondary antibody

The mouse monoclonal anti‐vinculin antibody was purchased from Sigma Aldrich (#V9131). Alexa Fluor 555 conjugated donkey anti‐mouse secondary antibody was obtained from Life Technology (#A31570).

HeLa cells (American Type Culture Collection) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX and pen-strep in 24-wells plates on coverslips. Fluorescein isothiocyanate (FITC)-labelled Stx1B (10 μg/mL in 300 μL of fresh medium) was added to the cells and incubated for 1 h on 37°C. The cells were then washed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 15 min and permeabilized with 0.1% triton X-100 in PBS for 10 min. Non-specific staining was blocked with 3% BSA in PBS for 1 h. Golgi apparatus (GA) was labelled with mouse monoclonal anti-human Golgin-97 primary antibody (0.4 μg/ml in 3% BSA for 1 h, Life Technologies, CA, USA) and with Alexa Fluor 555-conjugated donkey anti-mouse secondary antibody (1:1000 in 3% BSA for 1 h, A-31570, Life technologies). Coverslips were mounted with ProlongGold Antifade reagent with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). Immunostained cells were visualized with LSM-710 confocal microscope (Carl Zeiss, Germany), and images were acquired and processed using ZEN 2010 B SP1 software (Carl Zeiss).