Confocal immunofluorescence (IF) microscopy of human cells expressing DIII-RIgE fusion proteins. HEK293 cells were transfected with pcDNA3.1 plasmid expressing DIII-DENV1-RIgE or DIII-WNVKun-RIgE, and analysed by fluorescence imaging at 48 h posttransfection without cell permeabilization. Plasma membrane was stained with red-fluorescent WGA, nuclei with blue-fluorescent Hoechst 33342, and DIII-RIgE fusion proteins immunodetected with monoclonal anti-His6 tag antibody and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody. (A), Control cells transfected with empty pcDNA3.1 plasmid. (B), Cells transfected with pcDNA3.1-DIII-DENV1-RIgE plasmid. (C), Cells transfected with pcDNA3.1-DIII-WNVKun-RIgE plasmid. Panels (a) show views from the apical pole; panels (b) show the corresponding sagittal cell plane reconstructed from Z-stack images. Note that DIII-DENV1-RIgE and DIII-WNVKun-RIgE proteins were accessible at the surface of nonpermeabilized cells, and that they localized in membrane microdomains different from WGA-reacting regions.