Fig. 5

Morphological differences between subtypes of retinal macrophages. A–C Sholl analysis of resident (YFP⁺tdTomato⁺) and monocytic (YFP⁺tdTomato⁻) macrophages in WT (Arr1^+/+) and KO (Arr1^−/−) fate mapping (Cx3cr1^{+/YFP−CreER} Ai9^tdTomato post-tamoxifen) flatmount retinas. WT resident microglia in the IPL were larger, based on the number of intersecting radii (A gray bar), and more complex, based on total intersections and the ramification index (B, C), than KO resident microglia (orange) or KO monocytic macrophages (green) after 20 days of degeneration. Insets show representative morphologies of WT resident (gray), KO resident (orange), and KO monocytic (green) cells. n ≥ 25 cells per group over at least 3 fields of view. D After degeneration 11% of all resident macrophages (YFP⁺tdTomato⁺, yellow) and 60% of all monocytic macrophages (YFP⁺tdTomato⁻, blue) express detectable levels of MHCII, with the majority of these MHCII^high cells being monocyte-derived (mean ± SE; n = 8 fields of view/z-stack, 341 cells in total). E Representative maximum intensity projection through the IPL of a KO fate mapping retina after 20 days of light, labeled for all macrophages (YFP, green), resident macrophages (tdTomato, orange), and MHCII (blue). Cells with detectable levels of MHCII immunostaining were categorized as MHCII^high while those with undetectable levels were categorized as MHCII^low since we could not be sure based solely on IHC that cells were MHCII negative. F–H Sholl analysis revealed that KO MHCII^low resident microglia morphologically resemble WT resident microglia while MHCII^high resident microglia are morphologically similar to monocytic macrophages based on the number of intersecting radii (F), total number of intersections (G), and ramification index (H). n ≥ 3 fields of view / z-stacks per genotype, resulting in 7–55 cells per group. **p < 0.01, ***p < 0.001