For lung staining, the mouse lungs were filled with 2% low-melt agarose (Bio-Rad) through the trachea before dissection, chilled down in ice-cold PBS, and fixed in 4% paraformaldehyde (PFA) for 2 hours. For sectioning, agarose-filled lungs were embedded in 2% low-melt agarose and sectioned by Vibratome (Leica VT1000 S) into 200-μm-thick slices before staining. In the studies to compare the pulmonary lymphatic density in CCSP × VEGFC with WT × VEGFC mice, transverse sections from the part near the left bronchial branch of the left lobe were used for staining. In the study to compare the pulmonary lymphatic density in the lungs of control and 4T1-injected mice, transverse sections were made at the position of the detected nodules and corresponding positions in the control lungs. For the lungs without visible nodules on the surface, sections from the part near the left bronchus of the left lobe were used. For lacteal staining, approximately 1 cm of small intestine after the pancreatic curve was dissected, and the tube was cut longitudinally and pinned down (inside up) in silicone gel–coated six-well plates and fixed in 4% PFA for 2 hours at 4°C before staining. For liver staining, the right lobe of the mouse liver was dissected and fixed in 4% PFA for 2 hours at 4°C. Longitudinal sections (200 μm) were cut by Vibratome (Leica VT1000 S) before staining. Lymph nodes and other organs were dissected, embedded in OCT (optimal cutting temperature) compound, snap-frozen, and stored at −80°C until preparation of cryosections (7 μm).

For immunofluorescence stainings, sections were fixed with 4% PFA for 20 min, rinsed with PBS, and blocked in PBS with 0.2% bovine serum albumin, 5% donkey serum, and 0.3% Triton X-100 (blocking solution). Primary antibodies suspended in the blocking solution were incubated at room temperature for 2 hours or at 4°C overnight, followed by extensive washing and incubation with secondary antibodies. The antibodies used were Thy1.2 (rat, eBioscience), cytokeratin (wide spectrum, rabbit, Dako), LYVE-1 (rabbit, AngioBio), αSMA-Cy3 (mouse, Sigma-Aldrich), VEGFR3 (goat, R&D Systems), MECA-32 (rat, BD), RFP (rabbit, Rockland), podoplanin (goat, R&D Systems), and CD31 (rat, BD Pharmingen).

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