Fitc conjugated goat anti rabbit igg h l

Immunofluorescence assay was performed in BHK-21 cells that were plated on glass slides. The cells were infected with DENV2-16681, ZIKV or VacDZ and then incubated in a 37 °C incubator with 5% CO₂. Two days after infection, the cells were fixed and permeabilised with methanol at -20 °C. For immunofluorescence staining, the primary antibodies were mouse monoclonal anti-DENV/ZIKV NS1 protein (DN2, Abcam) at a dilution of 1:10, and rabbit monoclonal anti-ZIKV envelope protein (Ab00812-23.0, Absolute antibody) at a dilution of 1:200. Although DN2 is described as an anti-DENV NS1 protein antibody, we have found that it is cross reactive with ZIKV as well (Supplementary Figure 3). Secondary antibodies were FITC-conjugated goat anti-rabbit IgG (H + L) (F-2765, Thermo Fisher Scientific) at a dilution of 1:500 and Alexa 594-conjugated goat anti-mouse IgG (H + L) (A-11005, Thermo Fisher Scientific) at a dilution of 1:500. The slides were then mounted on microscope slides using Fluoroshield with DAPI (Sigma-Aldrich). Images were taken with an Olympus IX81 fluorescence microscope equipped with a UPlanApo 100x microscope objective lens (numerical aperture 1.35, Olympus) and Photometrics CoolSnap HQ CCD camera. Image acquisition was performed with MetaMorph software for Olympus.