Goat serum

BJ and HUVEC cells, fixed in p-formaldehyde (4% v/v in PBS; Lonza; Basilea, Swiss), were permeabilized with Triton X-100 (0.5% v/v in PBS; Lonza; Basilea, Swiss), blocked with goat serum (20% v/v in PBS; Lonza; Basilea, Swiss). Next, cells were incubated O/N at 4 °C with antibodies against VEGF (rabbit polyclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), αSMA (rabbit polyclonal, 1:100; Cusabio Life Science, College Park, MD, USA), VE-cadherin (mouse monoclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), FAP1α (rabbit polyclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), fibronectin (mouse monoclonal, 1:100; Abcam, Cambridge, UK), Col1A (mouse monoclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), FGF-2 (rabbit polyclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), vimentin (mouse monoclonal, 1:250; Santa Cruz Biotechnologies, Dallas, TX, USA), and vinculin (mouse monoclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA). F-actin was evaluated by 5 μg/mL of Phalloidin-FITC (Sigma-Aldrich; Saint Louis, MO, USA) for 30 min, at RT in the dark. The staining with anti-mouse and anti-rabbit antibodies and the nuclei and the confocal analysis were performed as described in [17 (link)].

EVs were labelled with BODIPY® FL N-(2-aminoethyl)maleimide. Briefly, 10 μL of Bodipy dye was added to 13.5 mL of S. dominica HR conditioned medium and mixed for 30 sec. After 5 min incubation at room temperature, EVs were isolated by ultracentrifugation as described before. After 24 and 48 h EV treatment, MIA PaCa-2 cells were harvested and used for confocal analysis performed as reported in a previous work^{70 (link)}. Briefly, cells were fixed in p-formaldehyde (4% v/v in PBS; Lonza; Basel, Switzerland), were permeabilized with Triton X-100 (0.4% v/v in PBS; Lonza; Basel, Switzerland), blocked with goat serum (20% v/v in PBS; Lonza; Basel, Switzerland) and then incubated with anti-annexin A1 antibody (rabbit polyclonal; 1:100; Thermo Fisher Scientific; Waltham, MA, USA), overnight at 4 °C. After two washing steps, cells were incubated with anti-rabbit AlexaFluor 555 (1:500; Thermo Fisher Scientific; Waltham, MA, USA) for 2 h at RT in the dark. To detect the nuclei, 4′,6-diamidino-2-phenylindole (DAPI, 1:1000) was used. Samples were vertically scanned from the bottom of the coverslip with a total depth of 5 μm and a 63X (1.40 NA) Plan-Apochromat oil-immersion objective. Images and scale bars were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH) and presented as single stack. Confocal microscopy analyses were carried out in three independent experiments.