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. 2020 Aug 24;147(16):dev185140. doi: 10.1242/dev.185140

Fig. 4.

Fig. 4.

Fgfbp1iEC-KO mice show delayed BBB maturation. (A-F′) Immunofluorescence analysis of BBB permeability to Cadaverine-AlexaFluor 555 in the cortex of WT (A,D) and Fgfbp1iEC-KO (B,C,E,F) P14 pups. Leakage of Cadaverine-Alexa555 into the cortical parenchyma was assessed 2 h after intraperitoneal injection of tracer (red; D-F′). Sections (100 µm thick) were also stained for claudin 5 (green; A-C′) to correlate tracer leakage with TJ integrity. Fgfbp1iEC-KO pups have increased BBB permeability in the cortex. C′ and F′ show magnifications of the boxed area in C and F, respectively. (G) Quantification of BBB permeability to Cadaverine-Alexa555 in the cortex of WT and Fgfbp1iEC-KO P7, P14 and P21 pups. Each dot represents the average fluorescence intensity of the tracer from confocal stacks of 4-9 cortical regions with either low or high BBB permeability per animal. Data are mean±s.e.m. and fold change compared with wild-type mice. *P<0.05; Student's t-test. Fgfbp1iEC-KO pups have increased BBB permeability at P7 and P14. (H-J‴) Analysis of BBB permeability and collagen IV deposition in the BM of cortical vessels in WT (H-H‴) and Fgfbp1iEC-KO (I-J‴) P14 pups. Leakage of Cadaverine-Alexa555 into the brain parenchyma was assessed 2 h after injection of the tracer in the peritoneum (H-J, red; dashed line in J). Sections (100 µm thick) were stained for podocalyxin (blood vessels, magenta; H′,I′,J′) and collagen IV (green; H″,I″,J″). Fgfbp1iEC-KO pups have increased BBB permeability in areas lacking collagen IV in the BM (dashed white line). (K-M‴) Analysis of BBB permeability, serum IgG leakage or Plvap expression in the cortex of WT (K) and Fgfbp1iEC-KO (L,M) P14 pups. Sections (100 µm thick) were stained for serum IgG to identify BBB leakage (magenta; K-M; dashed line in M), CD31 (red; K′-M′), Plvap (green; K″-M″) or merge (K‴-M‴). Plvap expression is higher in regions with leaky BBB and serum IgG extravasation. Dashed line indicates the area of BBB leakage. (N,O) Quantification of the number of IgG leakage foci (N) and Plvap expression (O) in the cortex of WT and Fgfbp1iEC-KO P7, P14 and P21 pups. Each dot represents a single animal. The number of foci with IgG leakage (i.e. high BBB permeability) is increased in Fgfbp1iEC-KO pups. Data are mean±s.e.m. *P<0.05, **P<0.005, ****P<0.00005; Student's t-test. Scale bars: 100 µm.