Fig. 3.

Immunostaining for specific markers in the gastrointestinal mucosa for the analysis of intestinal damage. Detection of (a). adherens junction proteins E-cadherin and β-catenin and (b). cellular polarity marker ezrin in the proximal and mid-small intestine following 48 h of infection with either S. aureus RN8098 WT, seb mutant, or seb complement. Tissues were collected from uninfected and infected mice and immunostained with E-cadherin (red) and β-catenin (green), or ezrin (green) and counterstained with DAPI (blue). Co-localisation of E-cadherin and β-catenin shows as yellow. Scale bar=100 µm. Detection of (c). cellular proliferation marker PCNA in the proximal small intestine following 48 h of infection with either S. aureus RN8098 WT, seb mutant, or seb complement. Proximal small intestine was collected from uninfected and infected mice and immunostained with PCNA (brown) and counterstained with haematoxylin (blue). Scale bar=100 µm. (d). Cellular proliferation was quantified by counting the number of PCNA positive and negative cells per crypt for 30 crypts per mouse, and is presented as the percentage of PCNA positive cells per crypt. Error bars=Mean± SEM; n=5–10 mice per group (WT=10; seb mutant=10; seb complement=9; uninfected=5). Crypt density was quantified by counting the number of crypts in a defined field of view for uninfected mice in comparison with fields of view from infected mice where histological damage was observed. At least 100 fields of view were counted per group (WT=146; seb mutant=126; seb complement=102; uninfected=100), and results are presented as the average number of crypts per field of view. Error bars=Mean± SEM; n=5–15 mice per group (WT=15; seb mutant=15; seb complement=9; uninfected=5). Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons test (**P≤0.01, ***P≤0.001).