Fig. 5: Validation of FZD7 localization in astrocyte endfeet.
From: Molecular profiling of brain endothelial cell to astrocyte endfoot communication in mouse and human
a Representative CUBIC-X expanded immunofluorescence images of FZD7, GFP-labeled astrocytes with endfeet, and lectin-labeled vessels in cortex of wild-type mice injected with AAV PHP-GfaABC1D-GFP. Expansion ratio (ratio measurement of astrocyte territory) = 1.8×. White squares indicate region of each image displayed at higher magnification below. Scale bars = 10 µm (overview) and 5 µm (inset). b Percent area of endfoot-GFP or lectin signal colocalizing with FZD7. Data shown as individual values, data points are shaded by mouse, and horizontal lines represent the median. LMM was performed to account for repeated measures per mouse: %Area~Marker + (1|Animal), N = 22 vessels from 4 mice, 5 or 6 vessel cross-sections/mouse; F(1,38.81) = 74.55. c Representative images of isolated cortical capillaries with endfeet (top) or endfoot depleted (bottom) and labeled for FZD7, AQP4 and lectin. Scale bar = 10 µm. Yellow line indicates position for pixel intensity profiles in (d, e). d, e Representative intensity profiles of FZD7 (magenta), AQP4 (blue), and Lectin (green) for isolated vessels with endfeet (d) and endfoot-depleted vessels (e). f Schematic of peak-to-peak distance measurement between peak intensities of FZD7 (magenta), AQP4 (blue) and Lectin (green). g Distance (µm) between peak intensities: Lectin-AQP4 (gray shades), Lectin-FZD7 (green shades), and AQP4-FZD7 (blue shades). Data are individual values, data points are shaded by mouse, and horizontal lines represent the median. LMM was performed to account for repeated measures per mouse: Distance~Marker + (1|Animal), N = 4 mice, 5 images/mouse; F(2,54) = 60.08, Tukey post hoc. h Schematic of intensity ratio metric (edge vs. center of vessel) for FZD7 (magenta), AQP4 (blue) and Lectin (green). i Intensity ratios of Lectin (green), AQP4 (blue), and FZD7 (magenta) in isolated vessels with endfeet and endfoot-depleted samples. Data are individual values, data points are shaded by mouse, and horizontal lines represent the median. LMM was performed on the indicated pairs to account for repeated measures per mouse: Intensity ratio~Condition + (1|Animal), N = 20 images/condition from 4 control and 5 depleted mice, 2-5 images/mouse. Lectin Ctrl vs. Depl, F(1,7.38) = 1.39; AQP4 ctrl vs. Depl, F(1,38) = 191.84; FZD7 ctrl vs. FZD7 Depl, F(1,38) = 54.85. Exact sample sizes per animal and source data are provided in a Source Data file.
