Marginated B cells are marked by CD49e, which mediates endothelial interactions. (A) Flow cytometry compared cell surface molecule expression on B cells obtained from nonperfused lungs (circulating blood remaining) with perfused lungs (circulating blood removed). (B) Flow cytometry expression of CD49e using a fluorescently conjugated anti-CD49e mAb. B cells were pregated for prototypical B1a, B1b, and B2 markers, and the percentage of CD49e-positive B cells is shown per group. FSC, forward scatter. (C) The expression of cell surface IgD was assessed by flow cytometry and compared between CD49e-positive B cells versus CD49e-negative B cells (A–C, n = 3 independent experiments using a total of six mice). (D) Intravital microscopy was used to visualize vascular B cells in mice treated i.v. with an inhibitory anti-CD49e mAb with or without an inhibitory anti-CD29 antibody compared with an appropriate isotype mAb control (pooled FOV replicates shown for n = 3 independent experiments using nine mice in total). (E) Intravital microscopy was performed with Cd19^ZsGreen1 and costained for IgD-positive B cells. Scale bar, 50 µm. (F and G) Vascular interaction times and track duration are shown as frequency distributions and pooled together to compare IgD-negative and IgD-positive B cells (n = 3 independent experiments with three mice in total). Exact P values were determined using Student’s t test or ANOVA. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Pooled data are presented as mean ± SD or box-and-whisker plots showing median and interquartile range.