Fig. 5. Internalized exogenous anti-CD63 antibody localized to the chlamydial inclusion. HEp-2 cells were infected with C. trachomatis E and cultured in the presence of exogenous anti-CD63 and anti-LAMP-1 antibodies (left panel), or anti-CD63 antibody and FITC-labeled dextran (right panel) from 24 hours to 48 hours post infection. Infected cells were then fixed and immunolabeled with anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 (left panel), or anti-mouse Alexa Fluor 594 only (right panel). TOPRO-3-labeling was used to identify intracellular bacteria and the host-cell nuclei. 0.5-µm-thick optical sections revealed that exogenously added anti-CD63 antibody trafficked to and accumulated in TOPRO-3-positive chlamydial inclusions (arrowheads). Concurrent addition of exogenous anti-LAMP-1 antibody and dextran-FITC to the culture medium resulted in their uptake into endocytic compartments of the host cell, but exclusion from the chlamydial inclusion. The arrow in the merged image indicates the position of the profile line used for analysis of intensity distribution. Bar, 20 µm. (Lower panels) Intensity distribution profiles confirmed the presence of anti-CD63 antibody (red line) within the TOPRO-3-positive inclusion (blue line). Anti-LAMP-1 antibody (green line, upper profile) and dextran-FITC (green line, lower profile) had high-intensity values peripheral to the inclusion and host-cell nuclei, but displayed background-intensity levels at the site of the chlamydial inclusion.