Fig. 3

Myeloid cell characterization after long-term peripheral myeloid cell engraftment. a–d Representative immunofluorescence images of myeloid cells in the brains of control (WT CON), irradiated (GFP-BM CON), and monocyte-engrafted (GFP-BM REPOP) mice stained for common myeloid marker ionized calcium-binding adaptor molecule 1 (IBA1, blue), microglial specific marker P2RY12 (red), and GFP (BM-derived cells, green) in the cortex (a–c) and hippocampus (d). Higher-resolution image showing P2RY12 immunoreactivity of IBA1⁺ and GFP⁺ cells (c). White dotted areas indicate pockets of high P2RY12 immunoreactivity, which coincide with a lack GFP⁺ cells. e–m Quantification of P2RY12, IBA1, and GFP⁺ staining: % area of P2RY12⁺ pockets, defined also as areas lacking GFP⁺ cell infiltrates (e–g), IBA1⁺ cell number per field of view (FOV) (h), P2RY12⁺IBA1⁺ cell number per field of view (FOV) (i), GFP⁺IBA1⁺ cell number per field of view (FOV) (j). For these measurements, images were captured and quantified in the hippocampus. k–m Quantification of IBA1⁺ cell morphology: average cell body diameter size (k), average process area (l), and process branching complexity (m). For these measurements, images were captured and quantified in the hippocampus. GFP⁺ staining was also used to distinguish between remaining microglia (IBA1⁺GFP⁻) and infiltrating monocyte (IBA1⁺GFP⁺) morphologies in GFP-BM REPOP mice. n Representative immunofluorescence images of myeloid cells stained for IBA1 (blue), macrophage/monocyte marker CD68 (red), and GFP (green). o Quantification of CD68 staining area coverage per FOV. p Quantification of TMEM119 staining intensity in the hippocampus. q Representative immunofluorescence images of myeloid cells stained for IBA1 (blue), CNS myeloid cell-specific marker TMEM119 (red), and GFP (green) in the hippocampus. r Quantification of the average mean intensity of TMEM119 in IBA1⁺ cells per FOV, comparing remaining microglia (remain) and infiltrating BM-derived cells (infilt). s Representative immunofluorescence images of proliferating myeloid cells stained for IBA1 (blue), phagocytic marker AXL (red), and GFP (green). t Quantification of the average mean intensity of AXL in IBA1⁺ cells per FOV, comparing remaining microglia (remain) and infiltrating BM-derived cells (infilt). Data are represented as mean ± SEM (n = 4–9). *p < 0.05, **p < 0.01, ***p < 0.001. CTX cortex, HC hippocampus, GFP green fluorescent protein, IBA1 ionized calcium adapter molecule 1. Scale bar ~ 150 μm (a); ~ 75 μm (b, d, n, q); ~ 60 μm (s); ~ 25 μm (c)