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Figure 5 Whole-mount confocal imaging of adult transgenic zebrafish hearts expressing lyve1:RFP (White: A and B) and lyve1:GFP (Green: D-G) to mark lymphatic vessels. Additional fluorescence signal from injected Firefli Fluorescent Blue-dyed Microspheres (MA; blue: A and B), Quantum dots (Qdot; magenta: A and B) and immuno-labeled neutrophils (Mpx; white: D-G) and Macrophage (IB4; red:D-G). (A) Intra-myocardial injection of 200 nm microspheres (cyan) and 10 nm Qdots (magenta) results in both being deposited at the injection site (magenta/cyan arrowheads). Qdots, but not MS are also highly concentrated within the lymphatic vessels within the lyve1:RFP positive endothelium (arrowheads, A; uninjected control, B). (C) Schematic representation of nanoparticle uptake by the cardiac lymphatic system. (D, n = 5) Uninjured zebrafish heart labeled with antibodies against GFP (lyve1:GFP) and Mpx and lectinB4 (IB4), showing relatively few Mpx-positive neutrophils or IB4-positive macrophage on the ventricle or BA, including within the lymphatic vessels on the BA (green arrowheads). (E, n = 4) 1 day post-cryo-injured (dpc) heart (mild injury) labeled at the same time shows a massive increase in Mpx-expressing neutrophils and more moderate increases IB4-positive macrophage including within the lymphatic vasculature (green arrowhead (GFP) white arrowhead (Mpx) red arrowhead (IB4). Mpx-positive cells are concentrated along the visibly darker blood vessels (inset, blue arrowhead) away from the wound site (orange arrowhead). (F, n = 5) 7dpc heart showing persistent elevation of Mpx-positive and IB4-positive cells on the ventricle and BA, where they are associated with lymphatic vessels. (G) High resolution imaging of lyve1:GFP -positive vessels (green arrowhead, outlined in blue) on the BA of a 7dpc heart IB4-positive macrophage (red arrowhead) within the vessel and Mpx-positive neutrophil (white arrowhead) on the surface of the vessel (H) Graph showing quantification of increase in IB4-positive cells within the BA lymphatic vessels of 1dpc (n = 4) and 7dpc (n = 5) zebrafish hearts compared to those of uninjured hearts (n = 5). *p<0.05; **p<0.01, unpaired t-test, Mean and SD. Scale bars 200 μm (A-G) and 50 μm (right panels, D-F).