CCL21 immobilization on the basement membrane of initial lymphatics may facilitate DC adhesion at CCL21-rich sites and passage through dedicated portals. (a) Collagen IV staining in whole-mount preparations of the ear skin shows perforations within the basement membrane of initial lymphatics (arrows in boxed region). (b) CCL21 staining reveals that several, but not all, of these perforations (arrows) are associated with CCL21 puncta. (c) Treatment of skin cross sections with type IV collagenase digested the basal membrane of initial lymphatics, markedly reducing collagen on lymphatics. Membrane-bound CCL21was dissociated, leaving behind small perinuclear depositions (arrows). (d) In the presence of the calcium chelator EDTA, collagenase IV treatment did not disrupt collagen (left) and CCL21 (right). (e–h) 24 h after contact sensitization, skin whole mounts were triple-stained for LYVE-1, CCL21, and MHC-II. MHC-II⁺ DCs accumulated outside initial lymphatics (e). (f) Enlargement of the boxed region in e shows in three dimensions a DC which extended protrusions toward the initial lymphatic vessel and contacted two CCL21-rich puncta. Two other examples of DCs contacting CCL21 are shown (g and h). (i–l) Similar results were obtained when the skin of CD11c-EYFP mice was analyzed. Collectively, these findings suggest that CCL21 is secreted from intracellular stores inside LECs and is immobilized on the basal membrane (often near preformed portals), to promote DC adhesion and site-specific transmigration.