Dm6000 cfs confocal microscope

To visualize lipid rafts, A549 cells were subjected to raft staining (Vybrant Lipid Raft Labeling Kit, Waltham, MA, USA) on ice, fixed, and mounted on glass slides in Prolong Gold anti-fade reagent with DAPI (4′, 6-diamidino-2-phenylindole) (Invitrogen, Carlsbad, CA, USA). To detect surface bound or internalized virus, A549 cells were incubated with IAV (X-31) at an M.O.I. of 10. After indicated incubation, cells were fixed with 4% paraformaldehyde in 1× PBS for 30 min at 4 °C or RT. Then, cells were mixed with 0.5% BSA in 1× PBST with 0.3% Triton X-100 (blocking and permeabilization) for 1 h at room temperature followed by staining with mouse anti-NP-FITC conjugated antibody (dilution 1:250 in blocking buffer) for 90 min at room temperature. After staining, cells were washed with 1× PBS (3 times, 5 min each) and mounted in Prolong Gold anti-fade reagent with DAPI (Invitrogen). The slides were visualized using Leica DM6000 CFS Confocal Microscope.

Antibodies and dilutions used are summarized in the Supplementary Methods. Cells (ECFCs, HUVECs, and fibroblasts) were fixed in 4% paraformaldehyde, blocked with blocking buffer (PBS with 10% FBS; 30 minutes, RT), permeabilized (5 minutes, RT) with 0.2% Triton‐X‐100, and incubated with primary antibodies (overnight, 4 °C). After washing, cells were incubated with an appropriate secondary antibody, and nuclei were stained with TOPRO‐3 Iodide (1:500) (1 hour, RT). Washed cells were mounted in SlowFade Gold antifade mountant (Thermo Fisher Scientific) and visualized using a Leica DM6000 CFS confocal microscope with 3 main fluorochromes, Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647, and 12‐bit resolution images were captured with the Leica application suite (version 2.7.3.9723). Appropriate isotype controls were stained (Figure S3). Antibodies used are listed in Table S1.