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. 2021 May 17;4:587. doi: 10.1038/s42003-021-02104-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic of a linear 3D culture with edge (blue), superficial layer (black), and deep layer(cyan) marked with rectangles. Sample locations of regions of interest are marked with gray circles. b Regions of interest (ROIs) from confocal z-stack images from culture locations indicated in a (from top bottom: DAPI, βIII-Tubulin, GFAP and merge, scale bar 10 μm). Mean and standard deviation of % of ROIs with directionality (alignment) for βIII-Tubulin (c) and GFAP (d) stained images at locations specified on the x-axis. Polar plot histograms of the directions (−90° to 90° with 5° bin width) found in the ROIs with directionality (c, d) for βIII-Tubulin⁺ neuronal (e) and GFAP⁺ astrocytic processes (f) at different locations. Radial lines indicates angles between aligned processes and culture’s long axis, and concentric circles show numbers of ROIs with alignment angle within a bin range. Green (e) and red (f) shaded region shows the ±30° angle range from culture long axis. g Average gray value (black dots) with mean (red thick line), and standard deviation (red shaded area) of GFAP⁺ processes at different distances (indicated on the x-axis) from top surface at the center of culture. h Means ± standard deviations of the number of GFAP⁺ cells in the superficial layer (top 10 μm) and deep layer (15–25 μm deep). i Box plot of nucleus (DAPI⁺ objects) alignment angles at different locations indicated on the x-axis. j Normalized number of DAPI⁺ objects in superficial layer (top 10 μm) and deep layer (15–25 μm deep) at center locations. k Means (black filled circle) with standard deviations of nucleus bounding cuboid height (along z direction) at different distances from top surface at center locations. Same 60 ROIs for each location from three cultures (20 each) were analyzed for c and d. Among these ROIs, 27, 57, 21, and 19 ROIs showed βIII-Tubulin alignment, and 45, 53, 27, and 40 ROIs showed GFAP alignment at center superficial, center deep, edge superficial, and edge deep locations, respectively. These directional ROIs were analyzed in e and f. For g, h, j, same n = 6 (100 μm × 100 μm) confocal z-stacks (40 μm deep from top surface) from three cultures’ center locations (two each) were analyzed. For k, n = 90 DAPI⁺ objects for each distance from surface (from three cultures) were analyzed. In total, 30 DAPI+ objects were counted from each culture’s z-plane for each distance from surface in k. Asterisks indicate results of (j) paired-sample t-test and (c, d, i, and k) two-sample Kolmogorov–Smirnov test (*p < 0.05, **p < 0.01, and ***p < 0.001).