Figure 3
Use of reporter lines and antibodies to identify splenic macrophages in the red and white pulp. (A–D): Representative images show staining of CSFR1+ macrophages with Mac2, Mac3, F4/80 and anti-lysozyme M antibodies in the red and white pulp. The dotted line represents the marginal zone. (E–H) Quantitative analysis shows that abundant CSF1R+ cells are located in the red pulp of the spleen. Dual immunofluorescent staining showed that the majority of the CSF1R+ cells in the red pulp are Mac3+ (F, 83.91% ± 3.002) and F4/80+ (G, 85.77% ± 3.117) positive. In contrast, only 38.52% ± 1.449 of CSF1R+ cells are labeled with Mac2 (E) and 28.90% ± 5.995 are Lysozyme M+ (stained with anti-lysozyme antibody, clone EPR2994(2)) (H). I-L: CSF1R/Mac2 (I), CSF1R/Mac3 (J), CSF1R/F4-80 (K) and CSF1R/Lysozyme (L) staining shows a low density of CSF1R+ cells in the white pulp. (M–P) Representative images show staining of CX3CR1+ macrophages with Mac2, Mac3, F4/80 and anti-lysozyme M antibodies in the red and white pulp. The dotted line represents the marginal zone. Q-T: Quantitative analysis shows that most of the cells stained with macrophage antibodies in the red pulp are CX3CR1 negative. U-X: Abundant CX3CR1+ cells are located in the white pulp. Mac2 labels most of the CX3CR1+ cells (88.62% ± 2.087, U), whereas Mac3 (V) and Lysozyme M (X) label more than half of the CX3CR1+ cells. F4/80 does not stain any CSF1R+ (K), or CX3CR1+ (W) cells in the white pulp of the spleen. Scale bar: 50 μm.
