Age-Dependent Enterocyte Invasion and Microcolony Formation by Salmonella
Figure 5
Characterization of S. Typhimurium infected enterocytes.
(A) Immunostaining of C57BL/6 mice simultaneously infected with two genetically labelled S. 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) Co-immunostaining of the SCV marker Lamp1 (red) and S. 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. (C) Quantitative analysis of the percentage of S. Typhimurium microcolonies associated with Lamp1 staining. All detected microcolony positive cells in 3 sections obtained from 3 S. Typhimurium infected animals were analyzed at day 4 p.i. The results represent the mean ± SD. (D) Co-immunostaining for active caspase 3 (white, arrow) and Salmonella (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. (E) Quantitative analysis of the percentage of active caspase 3 positive enterocytes among S. Typhimurium-infected and non-infected cells. 20 image areas (Magnification ×20) of intestinal sections from 3 S. Typhimurium infected animals were analyzed at day 4 p.i. The results represent the mean ± SD.