Intracellular localization of the recombinant EGFP protein. (A) Co-localization of viral antigens and EGFP. Infected cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and processed for immunolabeling. The designation on the upper right corner indicates the localization of the heterologous protein (EGFP); (α-YF) corresponds to the same cells stained with a hyperimmune antiserum to YF virus proteins; (DAPI) represents DAPI-stained cell nuclei; (merge) co-localization assessed by spectral overlap (yellow in right down panel) of the images of this preparation. (B) Co-localization of EGFP and the ER compartment. Live infected cells were labeled with ER-Tracker Red (Molecular Probes) and fixed in 4% paraformaldehyde. (EGFP) localization of heterologous protein; (ER) cells labeled with ER marker; (DAPI) nuclei counterstained with DAPI; (merge) co-localization assessed by spectral overlap (yellow in right down panels) of the images of this preparation.