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Figure 2

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ZDB-IMAGE-191230-1351
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Figures for Hassan-Abdi et al., 2019
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Figure 2

Genetic depletion of microglia worsens the pathology in Tg(HuC-hTauP301L:DsRed) embryos. (A) Outline of microglia depletion experiments. Embryos were injected at the single cell stage with a solution of antisense morpholino oligonucleotide targeting pU.1 transcripts. At 5 dpf, injected embryos were incubated in Neutral Red solution to sort microglia-depleted embryos. (B) Dorsal views of the optic tectum of 5 dpf wild-type microglia-depleted (B2) and untreated live embryos (B1), following incubation in Neutral Red solution. (C) Dorsal views of the optic tectum of 5 dpf wild-type microglia-depleted (C2) and untreated fixed embryos (C1), labeled with L-plastin antibody. (D,E) Measurements of pro-inflammatory cytokines in the brain of 5 dpf wild-type embryos with (n = 6), or without (n = 3) microglia; and Tg(HuC-hTauP301L:DsRed) embryos with (n = 11), or without microglia (n = 7). Both relative expressions of IL-1β (D, p = 0.035) and IL-8 (E, p < 0.0001) display a significant increase in the brains of Tg(HuC-hTauP301L:DsRed) embryos without microglia cells, compared to their siblings with microglial cells. (F,G) Representative Western blots membranes of total protein extracts from 6 dpf Tg(HuC-hTauP301L:DsRed) embryos with (left) or without (right) microglia, hybridized with antibodies against human total Tau (total Tau) or human phosphorylated Tau at Ser396 residue (pTau) (F); and quantification of corresponding pTau/total Tau ratio (respectively, n = 4 and n = 4) (G, p = 0.01). The ratio of hyperphosphorylated hTau to total Tau protein is significantly increased in microglia-depleted Tg(HuC-hTauP301L:DsRed) embryos. (H–J) Dorsal views of the telencephalon of 6 dpf Tg(HuC-hTauP301L:DsRed; nlrc3-likest73/+) embryos (H) and Tg(HuC-hTauP301L:DsRed; nlrc3-likest73/73) embryos (I), labeled with an antibody directed against human phosphorylated Tau at Ser396 and Ser404 residues (PHF1); and quantification of corresponding PHF1/hTauP301L-DsRed signal ratio (respectively, n = 4 and n = 6) (J, p = 0.0485). The quantification of the signal ratio of hyperphosphorylated hTau protein on brain sections from Tg(HuC-hTauP301L:DsRed; nlrc3-likest73/73) mutant embryos devoid of microglia confirmed the significant increase of this ratio displayed in protein extracts from Tg(HuC-hTauP301L:DsRed) embryos microglia-depleted with morpholino. ***p < 0.001; **p < 0.01; *p < 0.05. Scale bar (B,C,H,I) = 50 μm.

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