Hoechst 33342 for nuclear staining

To determine the influence of α-MG on the presence of DENV-2 NS5 and nuclear translocation of NF-κB p65, HepG2 cells were grown on glass coverslips for 24 h and infected with DENV-2 in the presence or absence of α-MG or RV as described above. After incubation at 37 °C in a 5% CO₂ atmosphere for 24 h, the treated cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and incubated with a mixture of mouse anti-DENV-NS5 antibody and rabbit anti-NF-κB p65 at RT for 1 h. After washing with PBS, the cells were incubated with a mixture of Cy3-conjugated goat anti-mouse IgG (Invitrogen), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen), and Hoechst 33342 for nuclear staining (Invitrogen) at RT for 1 h. Fluorescence images were visualized using a laser scanning confocal microscope (LSM 800; Zeiss Microscopy, Jena, Germany).
To investigate the influence of α-MG on the presence of DENV-2 NS5 and nuclear translocation of NF-κB p65 during the early phase of infection, HepG2 cells were infected with DENV-2 and treated with 20 μM of α-MG. The infected cells were harvested at 0, 1, 3, 6, and 24 h post-treatment. DENV-2 NS5 and NF-κB p65 were detected as described above.

To assess the impact of KG extract or EPMC on DENV infection, cells cultured on glass coverslips underwent a series of steps. First, they were fixed with a 4% paraformaldehyde solution in PBS, followed by permeabilization using 0.2% Triton X-100 in PBS. Subsequently, the cells were incubated with the 4G2 antibody. After thorough washing, a mixture containing Cy3-conjugated goat anti-mouse IgG (Invitrogen) was applied, along with Hoechst 33342 for nuclear staining (Invitrogen). The resulting fluorescence images were visualized using a laser scanning confocal microscope (LSM 800, Zeiss, Jena, Germany).
To investigate the influence of EPMC on the presence of DENV-2 NS5 and the nuclear translocation of NF-κB p65, the cells were subjected to a similar procedure. They were incubated with a mixture comprising mouse anti-DENV-NS5 and rabbit anti-NF-κB p65 antibodies, followed by a combination of Cy3-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG, and Hoechst 33342^{18 (link)}.