CD63 accumulated within the chlamydial inclusion. HEp-2 cells were infected with C. trachomatis E for 48 hours and analyzed by indirect immunofluorescence. The cytosolic face of the inclusion membrane was immunolabeled with anti-IncA antibody (anti-rabbit Alexa Fluor 488, see Results for details) and infected cells were then labeled with anti-CD63 antibody (anti-mouse Alexa Fluor 594). TOPRO-3-labeling was used to identify both intracellular bacteria and the host cell nuclei. (Upper panels) A 0.5-μm-thick optical section shows labeled CD63 localized to TOPRO-3-positive intracellular bacteria within the confines of the IncA-positive inclusion membrane. The white line in the merged image indicates the position of orthogonal analysis. Bar, 20 μm. (Lower panels) An orthogonal view of the infected cell (height of combined Z-sections 9.2 μm,) confirmed CD63 localization to TOPRO-3-labeled intracellular bacteria within the depths of the chlamydial inclusion as defined by IncA-labeling.