Figure 3

TLR4 is localized extra-and intracellularly in presence of C. pneumoniae. (A) Representative CLSM images after binding of C. pneumoniae at type II cells. Cells were without permeabilization stained with anti-TLR4 antibody that was detected by a secondary antibody coupled to Alexa488 (green). TLR4 is expressed at the external cell membrane. In un-infected cells no TLR4 expression was seen. Transmission images with pseudo-color red was used to highlight the outline of the cells. (B) Representative CLSM images of type II cells after incubation with C. pneumoniae at 37°C. Cells were after permeabilization stained with anti-TLR4 antibody that was detected by a secondary antibody coupled to Alexa594 (red) and labeled for F-actin with Alexa488-conjugated phalloidin (green). Nuclear DNA was stained with DAPI (blue). TLR4 mobilization to the external cell membrane reached a peak value after 10–30 min after exposure to C. pneumoniae. Areas of overlap between red and green are yellow, the bar equals 10 μm. Shown are representative examples, three more experiments gave similar results.