Characterization of <i>S.</i> Typhimurium infected enterocytes.

<p>(<b>A</b>) Immunostaining of C57BL/6 mice simultaneously infected with two genetically labelled <i>S.</i> Typhimurium strains orally administrated at a ratio of 1∶1. Staining was performed simultaneously with fluorophore-conjugated anti-GFP (green) and anti-mCherry antibodies (red). Note the presence of equal numbers of both green and red bacteria in the intestinal lumen in (i). Note also that the anti-mCherry antibody cross-reacted to some extend with the epithelial brush border. Counterstaining with Dapi (blue). Bar, i = 50 µm; ii and iii = 10 µm; iv and v = 5 µm. (<b>B</b>) Co-immunostaining of the SCV marker Lamp1 (red) and <i>S.</i> Typhimurium (green). i represents the merged image, ii–iv single color channel images. v shows a three-dimensional reconstruction to illustrate the punctuate Lamp1 staining surrounding the SCV. Counterstaining with Dapi (blue). Bar, i = 5 µm. (<b>C</b>) Quantitative analysis of the percentage of <i>S.</i> Typhimurium microcolonies associated with Lamp1 staining. All detected microcolony positive cells in 3 sections obtained from 3 <i>S.</i> Typhimurium infected animals were analyzed at day 4 p.i. The results represent the mean ± SD. (<b>D</b>) Co-immunostaining for active caspase 3 (white, arrow) and <i>Salmonella</i> (red) in small intestinal tissue sections obtained at day 4 p.i. Image ii represents an enlarged part of image i as indicated. Counterstaining with E-cadherin (green) and Dapi (blue). Bar, i = 50 µm. (<b>E</b>) Quantitative analysis of the percentage of active caspase 3 positive enterocytes among <i>S.</i> Typhimurium-infected and non-infected cells. 20 image areas (Magnification ×20) of intestinal sections from 3 <i>S.</i> Typhimurium infected animals were analyzed at day 4 p.i. The results represent the mean ± SD.