Ab233387

On the 3rd day after SNT, the L3/L4 DRGs were collected and immediately frozen after the mice were sacrificed. The tissue was lysed in RIPA buffer containing phosphatase and protease inhibitors (Beyotime, Shanghai, China). A BCA protein assay kit (Beyotime) was used to determine the concentration of protein. The proteins were transferred to polyvinylidene fluoride membranes after gel electrophoresis and then incubated with primary antibodies at 4°C overnight after blocking with 5% nonfat dry milk in TBS + Tween. The primary antibodies were as follows: rabbit anti-IL-6 (1:1000, ab6672, Abcam), rabbit anti-c-Jun (1:1000, Cat. No. D151652, Sangon), rabbit anti-Cd44 (1:500, ab51037, Abcam), rabbit anti-Timp1 (1:1000, ab109125, Abcam), rabbit anti-Csf1 (1:1000, ab233387, Abcam), and β-actin rabbit mAb (1:1000, AC026, Abclonal, Wuhan, Hubei, China), and were processed with goat anti-rabbit IgG (1:4000, AS014, Abclonal) at room temperature for 1 h. The signals of target proteins and β-actin were detected by a chemiluminescence detection system (Tanon4600, Shanghai, China). Relative protein expression levels were normalized using β-actin as an internal reference, and the band intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).

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).