Figure 7
Specificity and staining patterns of macrophage markers during the phases of cardiac repair. CSF1R-EGFP reporter mice underwent non-reperfused myocardial infarction protocols. Dual immunofluorescent staining for GFP and macrophage antibodies (Mac2, Mac3, F4/80 ab111101, Lysozyme antibody clone EPR2994(2)) was performed to identify cardiac macrophages and to evaluate the sensitivity and specificity of various markers. Control (C) mouse hearts have a small population of CSF1R+ cells (arrows). Quantitative analysis shows that the density of CSF1R+ cells in the infarcted myocardium significantly increased after 24 h, and peaked after 7 days of permanent coronary occlusion. No significant increase in the density of CSF1R+ cells was noted in non-infarcted remodeling segments. (A,B) Although at the peak of the proliferative phase (7d), 99.6% of CSF1R+ cells were labeled with Mac2 (arrows), significant populations of CSF1R+ /Mac2- cells were noted during the inflammatory (24 h–3d) and maturation phase (28d) (arrowheads). (C,D) Mac3 also stained ~ 90% of CSF1R+ cells at the 7-day timepoint (arrows). However, during the inflammatory phase of cardiac repair (24 h-3d), many CSF1R+ /Mac3- cells were noted (arrowheads), and only ~ 40% of CSF1R+ cells were Mac3 positive. (E,F) On the other hand, LyzM staining was more sensitive during the inflammatory phase with 98.1% of CSF1R+ cells expressing LyzM at the 24 h timepoint (arrows). Significant populations of LyzM-negative CSF1R+ cells (arrowheads) emerged during the proliferative and maturation phase. G. F4/80 staining was absent in CSF1R+ cells (arrowheads) infiltrating the infarcted myocardium. (**p < 0.01, ***p < 0.001, ****p < 0.0001 vs. corresponding control, n = 4–5/group). Scale bar: 20 μm.
