FIG. 9.

Cell surface localization of the G^vsv protein and HRSV G and F proteins in virus-infected cells by confocal microscopy. (A) Single antibody incubations for individual detection of G, F, and G^vsv. Vero cells were infected with the engineered viruses or virus A2 and fixed with freshly dissolved 4% paraformaldehyde at 24 h postinfection. G, F, and G^vsv proteins were detected by incubation with anti-VSIV G (αG^vsv), anti-HRSV G (αG) or anti-HRSV F (αF) antibodies, followed by incubation with an Alexa-594 (red)-conjugated secondary antibody. A2-infected cells were, in addition, incubated with Hoechst stain to visualize nuclei (panels 1 and 4). Images were generated by sequential scanning for GFP expression (green) or Hoechst stain (blue) and expression of viral antigens (G, F, and G^vsv) (red). For each image, the antibody used is indicated in the lower left corner. Size bar, 20 μm. (B) Double antibody labeling for dual detection of F and G^vsv proteins. Vero cells infected with virus RSΔsh,g/G^vsv were fixed as described above and incubated with anti-HRSV F and anti-VSIV G antibodies simultaneously. The F protein was visualized with an Alexa-594 (red)-conjugated antibody, and G^vsv was visualized with an Alexa-350 (blue)-conjugated antibody. Images were generated by sequential scanning for GFP expression (green), F expression (red), and G^vsv expression (blue). The antibodies used are indicated. Panel 3 is a merged image of panels 1 and 2.