Image analysis.

Confocal images used to assess overlap of Cxcr4, GFP, tdT or tdT/Ki67 with F4/80⁺, Iba1⁺ or Tmem119⁺ cell populations were acquired at 1 airy unit in sequential scan mode using a 40x, 1.3 NA oil objective. Developmental expression of Cxcr4 and Cxcr4-GFP in microglia was assessed for ≥50 Iba1⁺ cells per mouse. Microglia were randomly selected in the cerebral cortex, ventral telencephalon, and diencephalon. Layering of Iba1⁺ microglia in the cortex of E16.5 Cxcr4-deficient mice and control littermates were evaluated in 2×2 tile-scan images captured at 1 airy unit with a 20x, 0.5 NA objective (810,000 μm²) using DAPI to allocate cortical layers. For a given image, the number of microglia per layer was expressed as percentage of all counted microglia. Contribution of Cxcr4-CreER-labeled cells to microglia during development was determined in Cxcr4_CreER/wt; R26_CAG-LSL-tdT mice by assessing counterstained tdT in at least 100 microglia per mouse with at least 25 tdT⁺ cells evaluated each in the cerebral cortex, ventral telencephalon, diencephalon, and spinal cord. F4/80 was used as microglia marker in embryos and Iba1 in perinatal and postnatal mice. Overlap of counterstained Cxcr4-GFP or tdT with Tmem119 or Iba1 in day 3 infarcts was determined by evaluating at least 50 stained cells per condition and mouse. The Ki67⁺ percentage of monocytes and microglia in day 3 infarcts was determined by assessing Ki67 signal in all tdT⁺ Iba1⁺ and all tdT⁻ Iba1⁺ cells per view-field (≥50 cells per mouse). Engraftment of monocytes in the brain of mice undergoing photothrombotic stroke was assessed in 3 ROIs in sections stained for GFAP, Iba1, and tdT. ROIs#1-#3 were placed as illustrated in Fig. 4a. All Iba1⁺ cells in a given 50,625 μm² ROI were assessed for tdT signal. The number of monocytes (Cxcr4-IRES-GFP⁺ Iba1⁺ or tdT⁺ Iba1⁺) per area of infarct was determined for Cxcr4cKO and Cxcr4-control mice by counting all positive cells in the infarct. The infarct area was determined by co-staining for NeuN in the same section. Cell counts were divided by the infarct area. Infiltration of tdT⁺ Iba1⁺ monocytes at day 1 after tMCAO was evaluated in a 40x view-field placed in the NeuN⁺ olfactory tubercle by a researcher unaware of the experimental group. ImageJ software was used for cell counting and area measurements ⁵⁰. For quantification of infarct volume, brains were serially cut and stained for NeuN in 240 μm intervals. Stained sections were imaged using LSM510 with a 10x objective and the tile scan option. The infarct area was measured by a researcher unaware of the experimental group using ImageJ. Infarct volume was calculated from the sum of the infarct areas x section interval. Microglia morphometric analysis was performed with MotiQ⁵¹, a custom-made plugin for ImageJ. MotiQ thresholder (v0.1.2) was used to create figures from immunofluorescences for the MotiQ analyser (v0.1.3). The 3D microglial ramification index is defined as: cell surface area/(4π*((3*cell volume)/(4π))^2/3.