Identification of lymphatic vessels in skull periosteum. (A) Confocal images of dura-removed skull after CUBIC tissue clearance from 2-mo-old Prox1-CreERT2;Ai14 (Prox1;Ai14) mice. Prox1;Ai14 endogenous fluorescence (red) without antibody staining is coupled with IHC staining of Lyve1 (green) to label lymphatic vessels, and OsteoSense (white) is used to label compact cortical bone. Purple boxes (i and ii) indicate regions shown at higher magnification. LYVE1⁺;Prox1⁺ tubular structures are observed on the periosteal surface. Scale bars: 500 µm (overview), 50 µm (zoomed panels). (B) Coronal view (as in D) of skull subregions. Note the lymphatic vessel at the periosteum surface but not in OsteoSense-labeled compact cortical bone or bone marrow. Scale bar: 50 µm. (C) Dorsal view (as in F) of enlarged purple boxed region in B. Arrowheads represent Prox1-positive signals (red) in Lyve1-labeled lymphatic vessel tubes (green). Scale bar: 20 µm. (D and H) Schematic diagram illustrating the coronal view of the specific skull subregion (highlighted in panel A) for 3D imaging and analyses. (E and J) Coronal view of the specific skull subregion after CUBIC clearing from Prox1;Ai14 mice. Prox1;Ai14 endogenous fluorescence (red) without antibody staining is coupled with IHC staining of Lyve1 (green) to label lymphatic vessels. Endomucin (white) or CD45 (white) is used to label bone marrow. Note the Lyve1⁺;Prox1⁺ lymphatic vessels in skull periosteum but not in bone marrow. Scale bar: 200 µm. (F and I) Schematic diagram illustrating the dorsal view of the reconstructed 3D image. (G and K) Dorsal view of a skull subregion from a cleared Prox1;Ai14 skull stained with Lyve1 (green) and endomucin or CD45 (white). Note a network of lymphatic vessels distributed along the periosteal surface. Scale bar: 200 µm. All images are representative of n = 4 independent mice per condition.