Early life antibiotics affect intestinal barrier function in vivo, particularly in the distal small intestine. (A) H&E staining of proximal and distal small intestine. Black triangles indicate vacuolated enterocytes and white triangles indicate nonvacuolated enterocytes. Scale bars: 100 μm. (B) Quantification of the number of vacuoles per villi in the distal small intestine. (C) Permeability assay assessed by FITC-dextran concentration in serum 4 hours after oral gavage. (D) Villi length in proximal and distal small intestine. (E) Crypt depth in proximal and distal small intestine. Immunohistochemistry of (F) proliferation markers Ki67 and (G) phosphorylated histone H3 in proximal and distal small intestine. Scale bars: 100 μm. (H) Serum concentration of IgG and IgA in control and antibiotic mix–treated pups at P20 compared with adult mice. Statistical analysis was performed by the Mann–Whitney test because data were not normally distributed when assessed by the (B) D’Agostino and Pearson normality test, the (C–E and G) 2-tailed unpaired t test or (H) 1-way analysis of variance with the Tukey multiple comparisons test. Error bars indicate (B) medians with interquartile range or (C–E, G, and H) means ± SEM. Levels of significance are indicated: ∗P < .05, ∗∗P < .01, ∗∗∗P < .0001, ∗∗∗∗P < .0001. (B–E and G) n = 9–12 pups per group and (H) n = 6–9 P20 samples per group and n = 1–3 adult samples.