Alexa 594 conjugated goat anti mouse igg h l

Mouse monoclonal antibodies against NPM1 (clone FC82291, B0556, Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199, Sigma-Aldrich), β-actin (A5441, Sigma-Aldrich) and rabbit polyclonal antibodies against GFP (60–011, BioAcademia, Osaka, Japan) and histone H3 (#9715, Cell Signaling, Danvers, MA, USA), were purchased commercially. Mouse monoclonal antibodies against NPM (clone 9–2.6)^{30 (link)} and G196^{32 (link)} were described previously. Rabbit polyclonal antibody against NPM1c was raised using a synthetic peptide corresponding to the C-terminal sequence of NPM1c type A (DLCLAVEEVSLRK). Horseradish peroxidase-conjugated goat F(ab’)₂ anti-mouse IgG (H+L) (#710–133, Rockland Immunochemicals, Limerick, ME, USA), goat anti-rabbit IgG (111–035–003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and Alexa 594-conjugated goat anti-mouse IgG (H+L) (A-11032, ThermoFisher Scientific, Waltham, MA, USA) were purchased commercially.

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.

The following commercial antibodies were used: mouse monoclonal anti-FLAG (M2, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-FLAG (60–031, BioAcademia, Osaka, Japan) and anti-GFP (60–011, BioAcademia); HRP-conjugated goat F(ab’)₂ anti-mouse (710–1332, Rockland Immunochemicals, Limerick, ME, USA) and goat anti-rabbit IgG (111–035–003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA); Alexa 488-conjugated goat anti-rabbit IgG(H+L) (A-11034, ThermoFisher Scientific, Waltham, MA, USA) and Alexa 594-conjugated goat anti-mouse IgG(H+L) (A-11032, ThermoFisher Scientific).