McCoy cells were infected with C. trachomatis serovar L2, fixed with methanol at 24 (A, C, and E) and 48 (B, D, and F) h p.i., and reacted with mAb against chlamydial MOMP (green) in combination with pAb against fragment 4 of PmpD (A and B), pAb against chlamydial PmpA (C and D), or mAb against chlamydial IncA (E and F) (red). PmpA is localized in the cytoplasm while IncA is secreted outside Chlamydia and localized to the inclusion membrane. The beta-barrel (transporter domain) alone and included in the ≈80 kDa protein, remains associated with the interior of the cell wall of Chlamydia resulting in staining of chlamydial RBs with pAb against fragment 4 of PmpD in a doughnut-like pattern. Note, that contrary to pAb against fragments 1, 2, and 3, pAb against fragment 4 do not stain unfixed chlamydial RBs (not shown). The photographs were made using a Leica SL confocal microscope.