Alexa fluor 568 goat anti rabbit igg antibody

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.

Immunofluorescence staining was performed as previously described (18 (link)). RAW 264.7 cells (1 × 10⁵) grown on four-well tissue culture slides were incubated at 37°C for 12 h. PNR (100 µg/mL) and IFN-β (1,000 U) were added to the cells, which were cultured in a CO₂ incubator at 37°C for 12 h. Then, the medium was discarded and the cells were washed with PBS and infected with H1N1 (MOI = 1) for 2 h (18 (link)). Next, cells were cultured in a CO₂ incubator at 37°C for 24 h. Cells were then washed (with PBS, three times) and fixed with paraformaldehyde (4%) and 1% Triton X-100 for 30 min at room temperature (18 (link)). After blocking, the fixed cells were incubated overnight with M2-specific antibody, washed three times with TBS, and incubated with Alexa Fluor 568 goat anti-rabbit IgG antibody (1:1,000; Life Technologies, Eugene, OR, USA). Next, the cells were incubated with DAPI for 10 min and observed under a fluorescence microscope (18 (link)).

RAW 264.7 cells (0.5–1.0 × 10⁵) grown on a four-well tissue culture slide were cultured at 37°C for 24 h. For pre-treatment, WPS (100 μg/mL) was added to cells, which were incubated in a CO₂ incubator at 37°C for 12 h. After WPS treatment, the medium was discarded, and cells were washed with PBS and infected with H1N1 (MOI = 1) for 2 h. After infection, the medium was discarded and cells were washed with PBS; the medium was then added to cells, which were incubated in a CO₂ incubator at 37°C for 24 h. For post-treatment with WPS, cells were infected with H1N1 (MOI = 1) for 2 h. After infection, the medium was discarded, and cells were washed with PBS. Next, WPS was added to cells, which were incubated in a CO₂ incubator at 37°C for 24 h. At 24 h post-infection, cells were washed with PBS three times and fixed with 4% paraformaldehyde for 30 min and 1% Triton X-100 at room temperature. After blocking, fixed cells were incubated with an M2-specific antibody overnight, washed with TBS three times, and incubated with Alexa Fluor 568-goat anti-rabbit IgG antibody (1:1000; Life Technologies, Eugene, OR, USA) for 1 h at 25°C. Next, cells were incubated with DAPI for 10 min and observed by fluorescence microscopy.