Computational protocol: Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers

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Protocol publication

[…] For 2D imaging, fluorescence images were acquired with a laser confocal microscope (Leica TCS-SP5) equipped with a water-immersion objective lens (×63). Confocal images were processed with the Fiji package of ImageJ (NIH). For 2D cell orientation analysis, individual astrocytes in z-projected images were measured for their elongation and orientation angles. These angles were further calculated relative to the brain surface. For 2D Sholl analysis, we used coronal sections with 12 μm thickness and examined branching complexity of astrocytes at a coronal plane which contained their nucleus. Series of confocal images were converted to 8-bit gray-scale images, and the astrocyte processes were manually traced with the use of the Simple Neurite Tracer plugin of Fiji. Traces were z-projected and analyzed with the Sholl Analysis plugin of Fiji.For 3D imaging, fluorescence images (1.48-μm intervals) were acquired with a laser confocal microscope (Leica TCS-SP5) equipped with an oil-immersion objective lens (×40). Confocal images were processed with Photoshop CS4 software (Adobe), the Fiji package of ImageJ, or FluoRender (University of Utah). For quantification of 3D-cell orientation, individual astrocytes were extracted with the 3D Object Counter plugin of Fiji based on the red (tdTomato) channel of confocal images from Glast-EMTB-GFP;Glast-CreERT2;Rosa-CAG-LSL-tdTomato mice. The 3D objects of tdTomato-labeled astrocytes were then analyzed with the 3D Suite plugin for measurement of object volume, surface area, compactness, sphericity, convex hull volume, convex hull surface area, solidity, and convexity. The 3D Ellipsoid Fitting plugin was then applied to fit a 3D ellipsoid to objects in a labeled image, and the major axis length, middle axis length, minor axis length, elongation, flatness, and orientation angles relative to the XY, XZ, and YZ planes of the fit ellipsoid were measured with the 3D Suite plugin. The orientation angles relative to the XZ and YZ planes were further calculated relative to the brain surface. After the ROI for the tdTomato object was defined, the microtubule structure within that ROI was extracted with the Image Calculator plugin by subtraction of the GFP signal outside the cell from the original image of the GFP channel. The volume and surface area of these automatically extracted 3D microtubule structures were measured, and they were analyzed directly with the 3D Sholl Analysis plugin of Fiji. We excluded fibrous and perivascular astrocytes from our analysis. […]

Pipeline specifications

Software tools ImageJ, FluoRender
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus