Alexa fluor 488 green

Indirect immunofluorescence assay was used to perform a rapid evaluation of antiviral activities of compounds against IAV infection. For immunostaining, the IAV-infected or control cells were fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT). Cells were blocked with 1% bovine serum albumin (BSA) for 60 min at RT and then incubated with a mouse monoclonal antibody against influenza nucleoprotein (1:500 dilution, Sino Biological, Beijing, China) at 4 °C overnight. After three washes with PBS, the cells were incubated for 1 h at RT with an anti-mouse IgG antibody conjugated with Alexa Fluor^® 488 (green) (Cell Signaling Technology, MA, USA) at 1:1000 dilution. Nuclei were counterstained using 50 μL of 4,6-diamidino-2-phenylindole (DAPI, 300 nM; Sigma-Aldrich, MA, USA). Immunofluorescence was captured using the Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany).

Cells that grew on coverslips (Thermo Fisher Scientific, #12‐545‐80) were fixed using 4% paraformaldehyde (Sangon Biotech, #E672002‐0500) for 30 min, permeabilized with 0.5% Triton X‐100 for 15 min and then blocked using 5% BSA for 1 h. Cells were then incubated with the indicated primary antibodies overnight at 4°C, washed thrice with PBST, followed by incubation with Alexa Fluor 488 (green) (Cell Signaling Technology, #4408S or #4412S) and 555 (red) (Cell Signaling Technology, #4409S or #4413S) secondary antibodies. Fluoroshield mounting medium with DAPI (Abcam, #ab104139) was used to counterstain DNA. Fluorescence was detected using a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Buffalo Grove, USA). The images were processed using PhotoShop software.

The PAMs in different treatment groups were fixed with 4% paraformaldehyde for 30 min, then washed three times by PBS, permeabilized with 0.3% Triton-X100 solution per well for 15 min at room temperature, blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature and then incubated with a mouse monoclonal antibody against the p30 protein of ASFV (1:3,000 dilution) at 4°C overnight. After being washed three times with PBS, the cells were incubated for 1 h at room temperature with a goat anti-mouse secondary antibody conjugated with AlexaFluor^® 488 (Green; 1:1,000 dilution, Cell signaling Technology, MA, United States). Nuclei were counterstained using 50 μl of 4,6-diamidino-2-phenlindole (DAPI, 300 nM; sigma-Aldeich; blue). Immunofluorescence was captured using the Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany). The fluorescence densities (blue fluorescence and green fluorescence) of each well were digitalized by ImageJ software. The negative control group was set to 100%, and the other groups were compared with the negative control group. The EC₅₀ value was determined by quantifying the protection rate of compound treatment and calculated by nonlinear regression function using GraphPad Prism 7.0 software (Su et al., 2021 (link)).