Alexa fluor 488 conjugated donkey anti rabbit 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.

16HBE cells were seeded onto poly-L-lysine-coated coverslips (Solarbio, China) and treated as previously described. At the indicated time, the cells were washed in PBS, fixed with 4% PFA (Solarbio, China) and permeabilized with 1% Triton X-100 in PBS. The cells were blocked with 5% BSA at room temperature for 1 h and then incubated with the primary antibodies in blocking solution against EV-A71/CV-A16-VP1 (1:1,000, Millipore, USA), Nectin1 (1:100, Novusbio, USA), Claudin4 (1:200, Abcam, USA), E-cadherin (1:500, Abcam, USA) and ZO-1 (1:200, Abcam, USA) overnight at 4°C. Next, cells were washed with PBS three times and then incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Abcam, USA), Alexa Fluor 647-conjugated donkey anti-mouse IgG (Millipore, USA) or Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Biolegend, USA) for 1 h at room temperature. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:4,000, Beyotime, China). Slides were mounted with antifade reagent (Solarbio, China) and observed using a laser-scanning confocal microscope (Leica, Germany). The images were captured and processed using Adobe Photoshop 7.0 software.