Thymic heparan sulfate is restricted to adventitial mesenchyme and pericytes. Flow cytometric analysis (A) and quantitation (B) of heparan sulfate expression by CD45⁺ thymocytes, CD31⁺ endothelium, EpCAM-1⁺ TECs, CD34⁻integrin α7⁺ pericytes, and CD34⁺integrin α7⁻ adventitial mesenchyme in postnatal day–10 (P10) WT thymus. Gray filled histograms indicate staining levels where no anti–heparan sulfate primary antibody was added. (B) Analysis of mean fluorescence intensity (MFI) of heparan sulfate (HS) on CD34⁻integrin α7⁺ pericytes (orange bar) and CD34⁺integrin α7⁻ adventitial mesenchyme (purple bar) after gating on HS-expressing cells. Data shown from 3 separate experiments with a total of mice. Error bars in panel B represent mean ± SEM. For analysis of data in panel B, multiple comparison analysis was achieved by a 1-way analysis of variance followed by Tukey’s posttest in GraphPad Prism to determine statistical differences. (C) Confocal images of thymus sections from a P10 WT mouse stained with anti-CD31 (blue), anti-HS (green), and anti-CCL21 protein (red). White dotted line indicates the CMJ. Scale bar denotes 20 μm. Images shown representative of 5 mice. *P < .05, ** P < .01, ****P < .0001. C, cortex; M, medulla.