Heparan sulfate mediates CCL21 presentation at sites of thymic exit. (A) Confocal image of a blood vessel in a WT postnatal day–10 (P10) thymus section stained with anti-CD31 (red) and either anti–heparan sulfate (green; upper images) or an antibody to detect Δ-heparan sulfate (green; lower images). (B) Confocal images as in panel A, but sections were treated with heparinase III (H’ase III) enzyme before antibody staining. Scale bars denote 20 μm. Images typical of 2 separate experiments involving at least 3 mice. (C) Flow cytometric analysis of heparan sulfate expression by pericytes and adventitial mesenchyme before (blue histogram and blue bar) and after (red histogram and red bar) heparinase III treatment. (D) Flow cytometric analysis of Δ-heparan sulfate expression by pericytes and adventitial mesenchyme before (blue histogram and blue bar) or after (red histogram and red bar) heparinase III treatment. Bar charts in panels C and D show mean fluorescence intensity (MFI) expression levels of heparan sulfate and Δ-heparan sulfate, respectively. Data from 3 experiments with a minimum of 8 mice. (E) Flow cytometric analysis of CCL21m-RFP chemokine presentation by adventitial mesenchyme (upper panels) and pericytes (lower panels) in digested P10 WT thymus samples before (blue line) and after (red line) heparinase III treatment. Gray histograms represent control staining where no chemokine was added. Data from 3 separate experiments and 11 mice. Error bars represent mean ± SEM. Paired Student t tests were performed for statistical analysis of data in panel E. ****P < .0001.