Alexa fluor 488 goat anti mouse antibodies

Vero E6 and Huh7 cells were cultured on coverslips in 6-well plates and treated with MβCD and cholesterol as described above. At 48 h p.i. with the wild-type SARS-CoV-2, cells were washed with PBS and fixed with 4% formalin (15–20 min), permeabilized with 0.3% triton X-100 (10 min) and blocked with 3% BSA. After washing with PBS, cells were probed with primary antibodies overnight at 4 °C (anti-spike protein monoclonal antibody; Cell signaling, catalog #99423; 1:500 dilution). After three washes with PBS, spike proteins were detected with Alexa Fluor 488 goat anti-mouse antibodies (Abcam, catalog #ab150113). For F-actin detection, cells were incubated with fluorescent phalloidin solution for 30 min (Abcam catalog #ab235137). Coverslips containing the cells were mounted on glass slides using aqueous mounting media. Cells were observed using phase-contrast and fluorescence (3D Cell Explorer, NanoLive SA, Tolochenaz, Switzerland) microscopy, and photographs were taken.

Cells seeded on the scaffolds were allowed to grow for 24 h followed by washing with PBS, and were subsequently fixed with 4% paraformaldehyde for 10 min. The fixed cells were then permeabilised for 20 min using 0.5% Triton X-100 (Scharlau, Barcelona, Spain) before blocking by immersion in 10% goat serum (Elabscience, Houston, TX, USA) for 1 h at 37 °C. HDF was incubated with the primary antibodies, rabbit anti-human collagen I antibody (1:300 dilution; Abcam, Cambridge, MA, USA) and mouse anti-alpha smooth muscle antibody (1:500 dilution; Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by secondary antibody incubation with Alexa Fluor 647 goat anti-rabbit (Abcam) and Alexa Fluor 488 goat anti-mouse antibodies (Abcam), both diluted to 1:1000, for 2 h at 37 °C. The cells were counterstained with DAPI (Thermo Fisher, Waltham, MA, USA) for 15 min to visualise the nuclei. Between each post-fixing step, the samples were washed with PBS containing 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using an A1R confocal laser scanning microscope (Nikon, Minato City, Japan).