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Figure 4 (A) Tg(mpeg1:eGFP) zebrafish larvae were infected intravenously at 5 dpf with n = 200 T. carassii or with PVP control. At 3 dpi, larvae received 2 nl 1.13 mM iCLICKTM EdU, at 4 dpi were separated in high- and low-infected individuals and were imaged after fixation and whole mount immunohistochemistry 6–8 hr later (30–32 hr after EdU injection, ~9 dpf). Larvae were fixed and treated with iCLICK EdU ANDY FLUOR 555 (Red) development to identify EdU+ nuclei and with anti-GFP antibody to retrieve the position of macrophages, as described in the Materials and methods section. Larvae were imaged with Andor Spinning Disc Confocal Microscope using ×10 and ×20 magnifications. Maximum projections of the head (left panels, red boxes) and trunk (right panels, blue boxes) regions of one representative individual in PVP control, low- and high-infected zebrafish. Images capture macrophages (green) and EdU+ nuclei (red). In the PVP control group, EdU+ nuclei and GFP+ macrophages only rarely overlapped (white arrows, 20x), indicating limited proliferation of macrophages. In high- and low-infected individuals, the number of EdU+ macrophages increased (white arrows, 20x), indicating proliferation of macrophages in response to T. carassii infection. Blue arrowhead in the head of low and high-infected larvae, indicates the position of the thymus, an actively proliferating organ at this time point. The identification of EdU+ macrophages (white arrows) was performed upon detailed analysis of the separate stacks used to generate the overlay images, and are provided in Video 2. (B–C) Corrected total cell fluorescence (CTCF) calculated in the head (B) and trunk (C) region of larvae described in A. Symbols indicate individual larvae (n = 4–5 per group from two independent experiments). * indicates significant differences to the PVP control as assessed by One-Way ANOVA followed by Bonferroni post-hoc test. (D) Tg(mpeg1:eGFP) zebrafish larvae were treated as described in A and the number of EdU+ macrophages in the trunk region of PVP, low- and high-infected larvae was calculated. Symbols indicate individual larvae (n = 5 per group from two independent experiments). * indicates significant differences to the PVP control as assessed by One-Way ANOVA followed by Bonferroni post-hoc test.