Figure 7
Figure 7. Neutrophils are attracted to infection foci, yet much less phagocytic than macrophages, in zebrafish larvae. (A-D) In vivo observations in a swimming larva injected with red fluorescent E coli in the caudal vein at 52 hpf. Blue, green, and red arrowheads, respectively point at macrophages, neutrophils, and erythrocytes. (A-D) are video-enhanced DIC images, (A′-D′) show the corresponding overlay of DIC and red fluorescence of the bacteria; all images are at the same magnification. (A,A′) 1.5 hours after injection, yolk sac circulation valley: 2 macrophages, one of which loaded with methylene blue, have already phagocytosed many bacteria, whereas 2 neutrophils nearby are free of them; the neutrophil marked by an asterisk at the border of its nucleus is also displayed 45 seconds later in the lower left inset in A, to show its fast motility. The inset in A′ shows 4 more macrophages farther in the valley, 2 of which also loaded with methylene blue. (B,B′): 2.25 hours after injection, CHT (close to the site of bacteria injection): 4 macrophages, one loaded with methylene blue, have phagocytosed many bacteria, whereas a neutrophil among them has none. (C,C′) 2.5 hours after injection, CHT/ventral fin junction: a neutrophil that phagocytosed bacteria; (D,D′) images of the same neutrophil 8 minutes later. In (A,A′,B) thin straight lines indicate picture elements captured at a slightly different focus. (E) SB staining of neutrophils at 3 dpf, 5 hours after microinjection of either PBS or E coli bacteria in the left inner ear. (F) Confocal fluorescence image of a portion of the left ear at 3 dpf, 5 hours after injection of green fluorescent (GFP-expressing) E coli in the ear cavity. After fixation, the peroxidase activity of neutrophils was revealed with Cy3-tyramide (red), then GFP by anti-GFP immunohistochemistry with Alexa Fluor 488 as fluorophore (green), and the nuclei were stained with DAPI (blue). Two macrophages are highlighted by their large amount of phagocytosed bacteria, whereas only some of the recruited neutrophils show a few small bacteria-containing phagosomes. The upper right inset shows a neutrophil in an other focal plane, with 4 obvious such phagosomes. e indicates ear; oe, otic epithelium; and sc, semicircular canal. Scale bars, 10 μm.

Neutrophils are attracted to infection foci, yet much less phagocytic than macrophages, in zebrafish larvae. (A-D) In vivo observations in a swimming larva injected with red fluorescent E coli in the caudal vein at 52 hpf. Blue, green, and red arrowheads, respectively point at macrophages, neutrophils, and erythrocytes. (A-D) are video-enhanced DIC images, (A′-D′) show the corresponding overlay of DIC and red fluorescence of the bacteria; all images are at the same magnification. (A,A′) 1.5 hours after injection, yolk sac circulation valley: 2 macrophages, one of which loaded with methylene blue, have already phagocytosed many bacteria, whereas 2 neutrophils nearby are free of them; the neutrophil marked by an asterisk at the border of its nucleus is also displayed 45 seconds later in the lower left inset in A, to show its fast motility. The inset in A′ shows 4 more macrophages farther in the valley, 2 of which also loaded with methylene blue. (B,B′): 2.25 hours after injection, CHT (close to the site of bacteria injection): 4 macrophages, one loaded with methylene blue, have phagocytosed many bacteria, whereas a neutrophil among them has none. (C,C′) 2.5 hours after injection, CHT/ventral fin junction: a neutrophil that phagocytosed bacteria; (D,D′) images of the same neutrophil 8 minutes later. In (A,A′,B) thin straight lines indicate picture elements captured at a slightly different focus. (E) SB staining of neutrophils at 3 dpf, 5 hours after microinjection of either PBS or E coli bacteria in the left inner ear. (F) Confocal fluorescence image of a portion of the left ear at 3 dpf, 5 hours after injection of green fluorescent (GFP-expressing) E coli in the ear cavity. After fixation, the peroxidase activity of neutrophils was revealed with Cy3-tyramide (red), then GFP by anti-GFP immunohistochemistry with Alexa Fluor 488 as fluorophore (green), and the nuclei were stained with DAPI (blue). Two macrophages are highlighted by their large amount of phagocytosed bacteria, whereas only some of the recruited neutrophils show a few small bacteria-containing phagosomes. The upper right inset shows a neutrophil in an other focal plane, with 4 obvious such phagosomes. e indicates ear; oe, otic epithelium; and sc, semicircular canal. Scale bars, 10 μm.

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