Discriminating HSC-derived Macrophages from Microglia

The Cxcr4^CreER/WtR26^CAG-LSL-tdT model was used to discriminate HSC-derived macrophages from microglia^{32 (link)}. Adult Cxcr4^CreER/WtR26^CAG-LSL-tdT mice received tamoxifen (5 × 1 mg intraperitoneally) to label HSCs. Mice underwent 28 days tamoxifen washout before subsequent experiments. Recombination efficiency was assessed for circulating CD11b⁺CD115⁺ Ly6C⁺ monocytes by flow cytometry and exceeded 86% in all mice. Mice underwent 45 min MCAO and were processed for immunohistochemistry at days 1, 2, and 3 after MCAO induction (n = 3 each). HSC-derived macrophages were defined as tdTomato⁺Iba1⁺ and microglia as tdTomato⁻Iba1⁺-cells. Antibodies for co-staining in this model were: rat anti-RFP (Chromotek 5F8, 1: 500), goat anti-Iba1 (Abcam ab5076, 1: 500), rabbit anti-Osteopontin (Abcam ab63856, 1:2000), rabit anti-MCSF (Abcam ab233387, 1:2000), and rabbit anti-MMP12 (Abcam ab128030, 1:2000). Rabbit antibodies were detected using the biotin/tyramine amplification procedure and Streptavidin-AF488. Percent overlap of microglia and HSC-derived macrophages with M-CSF, Osteopontin, and MMP12 was examined in the striatal infarct in three 40x confocal view-fields per animal captured with an LSM900 microscope (Zeiss).