Microglial morphology

Confocal images were acquired from cortical layers 1, 3 and 5/6 in 20 µm (human tissue) or 10 µm (rat tissue) IBA1-stained sections using a 40 × objective and a z-stepsize of 1 µm (human) or 63 × objective and a z-stepsize of 0.1 µm (rat). IBA1⁺ cells were manually traced from 2D maximum intensity projections of the aforementioned confocal images using FIJI (NIH). Per image, around 10–20 cells were selected for tracing. Cells were randomly selected, but had to meet several inclusion criteria: (1) cells should be completely included within the z-stack borders of the image; (2) cells should not overlap with one another; (3) cells should not be associated to a vessel. Next, traced microglia were analyzed using the Sholl Analysis Plugin [17] with a 0.3 µm step size from the cell soma. Number of branches and junctions, and branch lengths were quantified by the AnalyzeSkeleton Plugin [1] using the same microglia cell tracings. Soma surface area of the traced microglia was measured using the freehand selection tool in FIJI.