S. flexneri interacts with and invades B lymphocytes upon ex vivo infection of human colonic tissue. (A and B) Fluorescence microscopy of histological analysis. T3SA− bacteria were found attached to the epithelium (A), whereas WT bacteria ruptured the epithelial barrier and got access to underlying tissue (B). (C) Confocal imaging of whole-mount tissue infected with WT bacteria, with top view (C) and orthogonal slice (C’). Reflection is shown in gray and crypts are outlined with a dashed line. (D and E) Confocal imaging of isolated lymph follicles in 150-µm-thick tissue sections, with top view (D) and orthogonal slices (E). Bacteria were stained with an antibody specific for S. flexneri 5a (red), B cells with anti-CD20cy (membrane receptor, green), and DAPI nuclei staining is shown in blue. Arrows point to bacteria. Bars: (A and B) 50 µm; (C and C’) 40 µm; (D) 20 µm; (E) 5 µm.