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Fig. 4. CD63-HA localizes to the chlamydial inclusion in transfected cells. HEp-2 cells were transfected with CD63-HA for 24 hours, then infected with C. trachomatis E for 48 hours and analyzed by indirect immunofluorescence and cryo immunoelectron microscopy. (Left panels) 0.5-µm-thick optical sections of infected cells immunolabeled with anti-HA antibody (anti-rabbit Alexa Fluor 488). TOPRO-3-labeling of the equivalent confocal slice was used to reveal intracellular bacteria and host-cell nuclei. Anti-HA antibody localized CD63-HA to TOPRO-3-positive intracellular bacteria in transfected cells (arrow) with no anti-HA antibody labeling associated with inclusions of adjacent untransfected cells (arrowhead). Bar, 20 µm. (Right panels) Transfected cells immunolabeled with anti-HA antibody (anti-rabbit-18 nm colloidal gold) revealed CD63-HA within the chlamydial inclusion and in compartments adjacent to the inclusion that morphologically resembled MVBs (arrows). Labeled CD63 was also evident in small vesicles within the inclusion lumen (lower panel, arrowhead; enlarged in insert). C, Chlamydia. The lack of label in mitochondria (m) served as an internal control for background labeling. Bar, 0.5 µm.





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