(a)
(b)
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Figure 6: Oxidative stress induction by ECwt infection of small intestinal villi. (a) Small intestinal villi were isolated from EC-infected mice ( per experiment) that had been treated or not with PGZ. The isolated villi were tested for the presence ROS at the indicated postinfection times using Cellomics® Oxidative Stress 1 HCS Reagent Kit. Quantitative detection of ROS was performed with DHE. Villus cells were costained with dihydroethidium (DHE) and Hoechst for quantitative detection of ROS and nuclei staining, respectively. Representative micrographs are shown. (b) Small intestinal villi isolated from mice were infected with ECwt and treated or not with PGZ. Villi were collected at the postinfection times indicated and subjected to the procedures indicated in (a). Representative micrographs are shown. (c) Fluorescence of ROS-positive cells at the indicated postinfection times was measured in randomly chosen triplicate fields per slide using the ImageJ program, and the corrected total cell fluorescence (CTCF) values are shown in terms of mean (SD) percentages of uninfected control mice. (d) Small intestinal villi were isolated from EC-infected mice that had been treated or not with PGZ. Villus cells were processed as indicated in (a) and the ROS-positive cells recorded using a fluorescence microscope (VanGuard). Data are expressed as mean percentage of ROS-positive cells through the 48 h.p.i. period. (e) Small intestinal villi isolated from mice were infected in vitro with ECwt and treated or not with PGZ. Villus cells were processed as indicated in (a) and the ROS-positive cells recorded as indicated in (d). Data are shown as mean ± SD percentage of ROS-positive cells through the 12 h.p.i. period. Villi treated with H2O2 were used as a control. Data are from three independent experiments performed in duplicate.