Prolong dapi mounting media

Immunofluorescence analyses were performed to visualize cytoskeleton in C- and CCM1-BOECs. To this purpose, a total of 50,000 cells were seeded on collagen-coated sterile coverslips (VWR international, Radnor, PA, USA) placed at the bottom of a 24-well plate. On the next day, cells were washed with PBS. Then, for staining actin filaments, cells were fixed, stained, and permeabilized in a single step by addition of 5 units/mL Alexa-546 phalloidin (Molecular Probes, Eugene, OR, USA), 100 μg/mL L-α-lysophosphatidylcholine, and 3.5% formaldehyde in cold PBS. Coverslips were mounted with Prolong+DAPI mounting media (Molecular Probes). Fluorescence images were taken using a confocal SP5 (DMI6000 CS Leica Microsystems, Wetzlar, Germany).
For tubulin/VE-cadherin staining, cells were fixed with 3.5% formaldehyde in PBS, washed, and blocked with 1% BSA in PBS for 1 h at 4 °C. Cells were incubated for 1 h at 4 °C with mouse antihuman tubulin/anti-VE-cadherin (1:100) (Sigma Aldrich). Following this, cells were washed thoroughly four times with PBS and incubated for 1 h at RT with goat antimouse IgG (H+L)–Alexa Fluor 568-conjugated antibody (1:200) (Thermo Fisher Scientific, Waltham, MA, USA). Finally, cells were washed with PBS and mounted and observed as described for actin.

The DAPI (4′,6-diamidino-2-phenylindole) staining method was used to detect the visual symptoms of apoptosis generated by ICI or Propranolol in HB cells.
Briefly, 5 × 103 HB cells were seeded on sterile, collagen-coated coverslips (13 mm diameter, VWR international, Radnor, PA, US) placed at the bottom of a 24 well-plate. On the next day, HB cells were treated with 100 µM Propranolol, or ICI for 24 and 48 h. Then, cells were washed with PBS and fixed with 3% PFA for 10 minutes at RT. After two PBS washing steps, samples were incubated mounted on glass slides using Prolong + DAPI mounting media (Molecular Probes, Eugene, OR, US). 40x confocal images were taken using the fluorescence confocal microscope Sp5 (DMI6000 CS Leica Microsystems, Wetzlar, Germany). FIJI-Image J software tool (NIH, MD, US) was used to identify and analyze the apoptotic nuclei.

Immunofluorescence analyses were performed to characterize the isolated BOECs using specific ECs marker VWF, and to stain for the pVHL (Novus, Oxon, UK). For this purpose, 5 × 10³ BOECs were seeded on sterile 13 mm diameter coverslips (VWR international, Radnor, PA, USA) placed at the bottom of a 24 well-plate, previously coated with collagen. On the next day, cells were washed with PBS and fixed with 3% PFA for 10 min at RT. After two PBS washing steps, samples were incubated with blocking solution (1% goat serum and 1% BSA, in PBS) for 1 h at RT. Then, cells were incubated overnight at 4 °C with rabbit anti-VWF antibody (1:50) (Abcam). Following this, cells were washed thoroughly four times with PBS and incubated for 1 h at RT with goat anti-rabbit IgG (H + L)-Alexa fluor 568 conjugate antibody (1:200) (Thermo Fisher Scientific). Finally, cells were washed with PBS and coverslips were mounted on glass slides using Prolong-DAPI mounting media (Molecular Probes), and 40× confocal images were taken using the fluorescence confocal microscope Sp5 (DMI6000 CS Leica Microsystems, Wetzlar, Germany). Green and blue channels represent VWF and DAPI stains, respectively.