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Fig. 3. Ultrastructural studies of the fin-wound response. (A,B) Whole-mount 3-dpf larvae prior to wounding that were histochemically stained with Sudan Black to reveal neutrophils (A) or immunostained with L-plastin antibody to reveal macrophages and neutrophils (B), indicating the absence of any leukocytes in the undamaged fin. (C) Scanning electron micrograph (SEM) of a 3-dpf zebrafish larvae fixed immediately post-wounding, indicating the site of lesion (arrow). (D,E) High-magnification views of the wound region at 0 minutes (D) and 10 minutes (E) post-wounding, indicating the rapidity of repair in control larvae. (F) Higher-magnification view of the boxed area in E shows complete epithelial sealing by 10 minutes. (G) Transverse resin section through a Methylene-Blue stained wounded larval tail region; red arrow indicates the route taken by leukocytes from the ventral vein to the site of wound damage in the ventral fin. (H) Transmission electron micrograph (TEM) transverse reconstruction of the entire fin, from the ventral vein (asterisk) to the damaged-fin tip (black arrow). The white arrow indicates fin epithelium. (I-M) False-coloured, high-magnification TEM images corresponding to various steps in the leukocyte migration route from the blood vessel towards the wound. (I) A neutrophil (purple) with characteristic electron-dense cigar-shaped granules is captured as it adheres to the vessel wall (green). (J) Once outside the vessel, the neutrophil changes its morphology, becoming more elongated and polarised as it follows chemotactic cues towards the wound (in the direction of the arrow). (K) Low-magnification TEM illustrating a number of neutrophils and a single macrophage (blue) at the wound site. (L) Illustrates a macrophage that has recently engulfed matrix debris. Areas circled with red indicate desmosomes at junctions between macrophages and adjacent cells at the wound site. The high-magnification inset shows engulfed collagen. (M) Illustrates a macrophage that has engulfed a cell corpse (arrow). Scale bars: A-C, 500 µm; D,E, 20 µm; F, 5 µm. G, 15 µm; H, 5 µm; I,J, 2.5 µm; K, 4 µm; L,M, 2 µm.
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