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. 2023 Sep 21;21(9):e3002308. doi: 10.1371/journal.pbio.3002308

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© 2023 Nualart et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 8 — (A-C) Immunofluorescence and confocal analysis and/or DIC imaging of the SCO (A) and dorsal ependymal cells (B). Staining of Frizzled-2 and SCO-spondin under normoglycemic conditions. Frizzled-2–positive immunoreactivity was mainly detected in the apex of ciliated cells (B). Scale bar: 20 μm. Quantitative analysis of Frizzled-2 immunoreactivity (C). The graph shows data from 4 biologically independent samples. The error bars represent the SD; ***P < 0.001, n.s. = not significant (one-way ANOVA with post hoc Tukey’s test). (D-F) Immunofluorescence and confocal analysis and/or DIC imaging of the SCO (D) and dorsal ependymal cells (E). Staining of Frizzled-2 and SCO-spondin under hyperglycemic conditions (CSF glucose concentration of 5 mM). Frizzled-2 immunoreactivity was detected in the apex of ciliated cells and on the apical membrane of ependymal cells (E). SCO-spondin and Frizzled-2 showed colocalization in cilia (E, DIC image, arrows). Scale bar: 20 μm. Quantitative analysis of Frizzled-2 immunoreactivity (F). The graph shows data from 4 biologically independent samples. The error bars represent the SD; **P < 0.01, ***P < 0.001, n.s. = not significant (one-way ANOVA with post hoc Tukey’s test). (G-J) Immunofluorescence and confocal analysis and/or DIC imaging of the SCO (G) and dorsal ependymal cells (H and J). Staining of Frizzled-2 and SCO-spondin under hyperglycemic conditions (CSF glucose concentration of 10 mM). Frizzled-2 immunoreactivity was detected mainly inside the cells (H and J, yellow arrows). Only a few cells showed immunoreactivity in the cilia and cytoplasm (J). Scale bar: 20 μm. (I) Quantitative analysis of Frizzled-2 immunoreactivity. The graph shows data from 4 biologically independent samples. The error bars represent the SD; **P < 0.01, ***P < 0.001, n.s. = not significant (one-way ANOVA with post hoc Tukey’s test). Scale bar: 20 μm. Data used to generate graphs can be found in S1 Data. BV, blood vessel; CSF, cerebrospinal fluid; DIC, differential interference contrast; d3v, dorsal third ventricle; SCO, subcommissural organ.