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. 2016 Nov 18;7:1829. doi: 10.3389/fmicb.2016.01829

Figure 8.

Figure 8

Morpholino knockdown of MyD88, verified by Western Blot analysis (A) of zebrafish MyD88 at 4 dpf, showing a depleted level of the protein in morphant fish. β-actin served as loading control. Survival rates of control and MyD88 depleted larvae at 36 hpi (B) show differences after intravenous injection of W. chondrophila. Morphant larvae show a slightly reduced survival rate compared to control larvae. Survival rates of 10 larvae for each condition were observed in three independent experiments, respectively. The bacterial load, determined by qPCR, (C) of MyD88 depleted larvae at 36 hpi is significantly (p = 0.0242) higher compared to control larvae. Statistical analysis was done by Student's unpaired t-test. Mean values ± SEM are shown by horizontal bars. Total numbers of neutrophils of whole individual control and MyD88 depleted larvae (D) were determined in the transgenic Tg(lyzC:dsRed)nz50 line at 36 hpi after intravenous injection of PBS or live W. chondrophila by counting dsRed expressing neutrophils after IF staining with an anti-dsRED antibody and subsequent CLSM analysis. The acquired 3D stacks of whole larvae were analyzed with the fluorescent spot counting tool in Imaris (Bitplane). The results show a significant depletion of neutrophils after injection of W. chondrophila compared to PBS injection in both normal and MyD88 depleted larvae with no significant differences between the two groups. Statistical analysis was done by one-way ANOVA with Bonferroni's posttest. ***p < 0.001, **p < 0.01, ns = not significant. Mean values ± SEM are shown by horizontal bars.