Figure 3. Immunofluorescence antibody staining of TLR4 in HCE cells and TLR4 transfected HEK-293 cells.

HCE or HEK-293 cells were grown to confluence on 4 well chamber slides. HCE cells were stimulated with A. castellanii trophozoites (1×10⁵ cells/ml) for 24 hours; HCE control cells and HEK-293 (positive control) cells were left untreated for 24 hours at 37°C. After the incubation period, cells were fixed with 4% paraformaldehyde, and then incubated with either PE anti-human TLR4 or PE Mouse IgG2α isotype control for 1 hour. To visualize the nuclei sections were counterstained for one minutes in 150 ng 4,6-diamidino-2-phenylindole, dilactate (DAPI). Three slides in each group were viewed using fluorescence microscopy. Images were captured with an Olympus AX70 Fluorescence Microscope. The results were expressed as percent of cells positive for TLR4 by counting the number of TLR4 positive cells divided by the number of live cells × 100. Cells that stain with DAPI were counted as live cells.