Detection of cHtrA protease in the cytosol of C. trachomatis -infected cells. HeLa cells infected with C. trachomatis L2 organisms were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue). The mouse antibodies included an anti-cHtrA (CT823) antiserum (raised with GST-cHtrA fusion protein) at various dilutions (A), the anti-cHtrA antiserum at 1:1000 dilution (B, panels a & f), mAb 6A2 (b & g, also raised with the GST-cHtrA fusion protein), mAb (100a) against CPAF (c & h), mAb (BB2) against IncA (d & i) and mAb (1L11C3) against HSP60 (e & j). The mouse anti-cHtrA staining (red) was also co-labeled with a rabbit anti-IncA antibody (green; C). Note that the anti-cHtrA antibodies detected signals both inside the chlamydial inclusions with (yellow arrowheads) or without (red arrowheads) overlapping with the chlamydial organisms and in the host cell cytosol (red arrows) while the anti-CPAF antibody mainly detected signals in the host cell cytosol.