Region-specific skull channels and bone marrow. (A) Representative images of parietal and interparietal bone sections from 2-mo-old mice, stained with antibodies against VEGFR3 (red) and Col1a1 (white). The white boxes indicate magnified regions of the overview images. The purple circles numbered 1 and 2 show the sampling areas for parietal and interparietal bones, respectively, as highlighted in B. Purple arrowheads indicate individual bone marrow channels. Scale bars: 500 and 200 µm (inset). (B) Representative images of meningeal vessels from 2-mo-old Prox1-eGFP mice (green). Purple circles 1 and 2 indicate the parietal and interparietal bone sampling regions. (C) Quantification of the number of channels connecting to the periosteum and meninges in 650-µm-thick skull samples. (D and E) Enlarged images of parietal and interparietal bones from 2-mo-old mice stained with VEGFR3 (red) and Col1a1 (white). The white boxes highlight regions within the skull, as shown in the lower overview images. Purple circles numbered 1 and 2 mark the parietal and interparietal bone sampling areas. Scale bars: 500 and 200 µm (inset). Imaris was used to colocalize color calculation function for volume quantification. We first created a mask of the ROI by setting thresholds on the channels of interest, then used the “Surface-Surface Coloc” extension to generate a surface object from that mask, and finally calculated the volume of that surface object within Imaris. All data are represented as the mean ± SEM calculated by Student’s t test, n = 4 mice, and three images were analyzed from each mouse, ***P = 0.0002 (interparietal bone) or 0.0004 (parietal bone) by two-way ANOVA. ROI, region of interest.