Immunofluorescence Analysis of Viral Infection

For the immunofluorescence analysis, we used a slightly modified version of the previously used immunofluorescence analysis method^{26 (link)}. Briefly, MDCK cells were cultured in 4-well tissue culture slides (5 × 10⁴ cells/well) for 16 h. Subsequently, H1N1 (MOI = 1) and A/PR/8/34-GFP (MOI = 10) were mixed with different concentrations of GHE (100 and 200 μg/mL) and GN (10 and 100 μM), and the mixtures were incubated at 37 °C for 1 h. MDCK and A549 cells were infected with these mixtures at 37 °C for 2 h. Thereafter, the virus was removed, and the cells were washed three times with PBS and were cultured in a CO₂ incubator at 37 °C for 24 h. The cells were then washed three times with cold PBS and fixed with 4% paraformaldehyde in PBS and 1% Triton X-100 for 10 min each at room temperature. After blocking, the fixed cells were incubated overnight at 4 °C with NP- and NA-specific antibodies, washed three times (5 min per wash) with TBS, and incubated with Alexa Fluor 568 goat anti-rabbit IgG antibody (1:1,000; Life Technologies, Eugene, OR, USA) and washed three times (5 min per wash) with TBS. Next, the cells were incubated with DAPI for 10 min and measured using fluorescence microscopy.