Zebrafish larvae (5 dpf), either (A) Tg(tnfa:eGFP-F x mpeg1:mCherry-F) (n = 8–13 larvae per group from four independent experiments), or (B) Tg(il1b:eGFP-F;mpeg1:mCherry-F) (n = 7–8 larvae per group from two independent experiments), were infected as described in Figure 7. At 4 dpi, larvae were separated in high- and low-infected individuals and imaged with a Roper Spinning Disk Confocal Microscope. Scale bar indicate 25 μm. (A) In non-infected PVP controls (left panel), several macrophages can be observed outside the vessel but none was positive for tnfa. In low-infected individuals (middle panel), macrophages were present inside and outside the vessel. Except the occasional macrophage showing tnfa-eGFP expression (white arrowhead), they generally did not exhibit strong eGFP signal. In high-infected individuals however, foamy macrophages (asterisks) as well as endothelial cells (bright green cells) or other leukocytes, were strongly positive for tnfa-eGFP expression. (B) il1b-eGFP expression was generally low in non-infected PVP controls. In low-infected larvae, il1b-positive macrophages were rarely observed (white arrowhead). In both high- and low-infected fish, some endothelium cells in the cardinal caudal vein show high il1b-eGFP expression (bright green cells in middle and right panel). In high-infected individual, however (right panel), foamy macrophages inside the vessel (asterisks) as well as other macrophages lining the vessel (white arrowhead) and leukocytes in the tissue (blue arrow), were positive for il1b-eGFP expression. (C) Quantification of the total number of foamy and non-foamy macrophages and of the number of those that are positive or not for il1b or tnfa. All foamy macrophages are positive for il1b or tnfa, and high-infected individuals have generally a higher number of il1b or tnfa positive macrophages than low-infected or PVP individuals. *, indicate significant differences as assessed by Two-Way ANOVA followed by Bonferroni post-hoc test.
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