Panel A includes plots for CD45, CD326, CD11c, F4/80, MHCII, Ly6C, and CD11b, highlighting Langerhans cells, macrophages, dendritic cells, and monocytes. Panel B shows flow cytometry plots of CD169 positive macrophages, CD169 negative MHCII positive macrophages, and CD169 negative MHCII negative macrophages, with a bar graph indicating the ratio of fixable viability dye (FVD) and DAPI staining. Panel C displays immunostaining of CD169, CD31, and DAPI in normal mouse back skin, highlighting blood vessels. Panel D shows immunostaining of CD169, LYVE1, and DAPI, highlighting lymphatic vessels. Panel E presents immunostaining of IBA1, CD169, and DAPI in mouse back skin after treatments with isotype mAb or anti-CSF1R mAb. Panel F includes a bar graph showing the number of IBA1 positive CD169 negative cells in the dermis per high-power field (HPF). Panel G shows a bar graph of the number of IBA1 positive CD169 positive cells in the dermal white adipose tissue (DWAT) and adventitia per HPF. The photos and diagrams together illustrate the identification and analysis of different macrophage subpopulations in mouse skin.
Skin CD169+ macrophages are CSF1R-dependent perivascular macrophages . (A) Gating strategy to identify skin LCs, macrophages (Mϕ), DCs, and monocytes (representative of five mice). (B) The ratio of FVD– DAPI– (orange), FVD+ DAPI– (aqua), and FVD+ DAPI+ (black) in CD169+ Mϕ, CD169– MHCII+ Mϕ, and CD169– MHCII– Mϕ (n = 5, data are shown with mean ± SD, data are representative of two independent experiments). (C) Immunostaining of CD169 (green), CD31 (red), and DAPI (blue) in normal mouse back skin. BV, blood vessels. (D) Immunostaining of CD169 (green), LYVE1 (red), and DAPI (blue) in normal mouse back skin. LV, lymphatic vessels. (E) Immunostaining of IBA1 (red), CD169 (green), and DAPI (blue) in mouse back skin after two treatments of subcutaneous injection of either isotype mAb or anti-CSF1R mAb. (F) The number of IBA1+ CD169− cells in the dermis per HPF was analyzed using Imaris (×20) (n = 4 for each group). P value was calculated using Student’s t test. (G) The number of IBA1+ CD169+ cells in the DWAT, and adventitia per HPF was analyzed using Imaris (×20) (n = 4 for each group). Each circle represents one mouse. P value was calculated using Student’s t test.