Alexa fluor 647 conjugated donkey anti mouse igg

Autophagosome formation in 16HBE cells with different treatments was examined by IF staining. At the indicated times, cells plated on chamber slides were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) (Solarbio, China) for 30 min at room temperature and were then rinsed with PBS. After permeabilizing with 1% Triton X-100 in PBS for 15 min and blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature, cells were incubated overnight at 4 °C with primary antibodies against EV71/CA16-VP1 (1:1000; Millipore, USA), LC3 (1:1000; Cell Signaling Technology, USA), TLR7 (1:1000; Abcam, USA) or M6PR (1:1000; Abcam, USA) diluted in a blocking solution. The next day, the cells were washed three times with PBS, and Alexa Fluor 647–conjugated donkey anti-mouse IgG (diluted 1:1000; Millipore, USA) and Alexa Fluor 488–conjugated donkey anti-rabbit IgG (diluted 1:1000; Biolegend, USA) were added to the cells, which were then incubated at 37 °C for 1 h in the dark and washed with PBS. Finally, the nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, 1:4000, Beyotime, China), and the slides were mounted with antifade reagent (Solarbio, China). The images were visualized and captured with a laser-scanning confocal microscope (Leica, Germany) using the appropriate filters.

HUVECs were seeded onto poly-L-lysine-coated coverslips (Solarbio, China). At the indicated time, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and permeabilized with 1% Triton X-100 in PBS for 15 min at room temperature. After blocking with 5% bovine serum albumin, the cells were incubated with the primary antibodies in blocking solution against EV71/CA16-VP1 (1:1000 dilution; Millipore, USA), Nectin1 (1:100 dilution), Claudin4 (1:200 dilution), VE-cadherin (1:500 dilution), and ZO-1 (1:200 dilution) overnight at 4 °C. Next, the cells were washed with PBS three times and then incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:1000 dilution; Abcam, USA) or Alexa Fluor 647-conjugated donkey anti-mouse IgG (1:1000 dilution; Millipore, USA) for 1 h at room temperature. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:4000 dilution; Beyotime, China). Following three washes in PBS, the coverslips were mounted on slides with an antifade reagent (Solarbio, China). Finally, images of the cells were captured with a laser-scanning confocal microscope (Leica, Germany) and processed using Adobe Photoshop 7.0 software.