Tcs sp5 laser scanning fluorescence microscope

ECs were nucleofected with a mCherry-vWF expressing plasmid [5 (link)] by using the Amaxa Nucleofector Technology (Lonza, Milan, Italy). Twenty-four h after nucleofection, cells were serum starved or not for 16 h in endothelial basal medium (EBM) containing 0.5% FBS alone or in combination with 3-MA (5 mM) (Sigma-Aldrich, St. Louis, MO, USA) or a pharmacological inhibitor of cathepsin-β, CA-074Me (50 µM) (Enzo life sciences, Farmingdale, NY, USA). Cells were treated for 1 h with 10 ng/mL of GST, p24 or p17 and fixed with 4% paraformaldehyde. When reported, p17 was preincubated for 30 min at 37 °C with 1 μg/mL of unrelated control mAb (Ctrl mAb) or p17-neutralizing mAb MBS-3. When indicated, serum-starved ECs were treated for 1 h with the PI3K inhibitor LY294002 (10 μM) or WT (100 nM) (Enzo life sciences, Farmingdale, NY, USA) before p17 stimulation. Fluorescence was analyzed using a Leica (Wetzlar, Germany) TCS SP5 laser scanning fluorescence microscope and the imaging software Leica Application Suite. The number of puncta per cell was quantified using ImageJ software, by counting vWF-positive puncta in 20 cells/experiment. Error bars represent the standard deviation calculated as the mean of 3 independent experiments with similar results.

Infected Vero E6 cells were seeded (5x10⁴ cells/well) in 8-well chamber slides (Becton–Dickinson, Franklin Lakes, New Jersey, USA). Twenty-four h later, cells were fixed with 2% paraformaldehyde in PBS for 10 min, permeabilized with 0.1% Triton X100 in PBS, and saturated with 3% BSA, 0.1% Tween 20 in PBS. For staining, cells were incubated overnight with a human serum containing IgG to SARS-CoV-2 (1:200 dilution) followed by Alexa Fluor 488-conjugated anti-human IgG (Thermo Fisher Scientific). Nuclei were counterstained with 4′,6-diamidino,2-phenylindole (DAPI, Merck). Cells were analyzed using a Leica (Wetzlar, Germany) TCS SP5 laser scanning fluorescence microscope and the imaging software Leica Application Suite.

Cells treated with GST- or GST-ARV p17 were fixed, permeabilized, blocked with 3% BSA and incubated overnight with a rabbit polyclonal antibody to ARV p17 (dilution of 1:100; Abcam). Then, the cells were washed and incubated with AlexaFluor 488 conjugated goat anti-rabbit IgG secondary antibody (dilution of 1:500; Thermo Fisher Scientific) for 1 h at room temperature. After three washes with PBS, slides were mounted and samples were observed using a Leica (Wetzlar, Germany) TCS SP5 laser scanning fluorescence microscope and the imaging software Leica Application Suite.