FIG. 2.

Immunofluorescence analysis of cells infected with the B1940 cps mutant or B1940. HeLa cells were infected with the B1940 cps mutant or B1940 as shown. Images were taken after gentamicin treatment for the B1940 cps mutant (A, A′, and A′′) or after gentamicin treatment (B, B′, and B′′) or 8 h after gentamicin treatment (C, C′, C′′) for B1940. To distinguish between extracellular and intracellular bacteria, the anti-N. meningitidis antibody (α-N. meningitidis) and its secondary antibody were used before (OUT) (A, B, and C) or after (IN + OUT) (A′, B′, and C′) permeabilization of cells with saponin. The secondary antibody used before permeabilization was Cy5 conjugated, while the one used after permeabilization was fluorescein isothiocyanate conjugated. To detect a cellular marker, we used anti-Lamp1 followed by a tetramethyl rhodamine isothiocyanate-conjugated secondary antibody. Merged images of different channels are shown in A′′, B′′, and C′′. Bars, 10 μm.