Lyve1^hi membrane-associated macrophages are located in serous membrane of tissue parenchyma. (A) Whole-mount, confocal microscopy tile-scan reconstructions to examine the location and distribution of LYVE1^hi mesenteric macrophages in tamoxifen-induced Prox1^CreER: R26^LSL-tdTomato mice (red, Prox1-expressing lymphatic collector; white, LYVE1-expressing macrophages and lymphatic capillaries; blue, nuclei). Scale bar, 400 µm. Images are representative of two independent experiments with scanning of a large region of tissue. (B) Schemes for Tomato⁻ BM transplantation to whole-body-irradiated mice (created with BioRender.com). Sorted CD45.2 Tomato⁻ BM cells from Lyve1^cre:R26^LSL-tdTomato mice were transplanted to irradiated congenic CD45.1 WT mice. Tomato reporter will label only adult macrophages with an active LYVE1 promoter in adulthood, bypassing embryo-restricted activity at this promoter. (C) Histogram showing Tomato reporter expression of tissue-resident macrophages in brain, spleen, lung, and gut mesentery of Tomato⁻ BM transplanted CD45.1 recipient mice. (D) Quantification of Tomato expression in donor-derived tissue-resident macrophages of Tomato⁻ BM transplanted CD45.1 mice. (C-D) Data are pooled from at least two independent experiments (n = 6, mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001. (E) Whole mount images of the mesenteric membrane in Tomato⁻ BM transplanted chimeric mice (red, Tomato⁺ macrophages; blue, collagens imaged by second harmonic generation [SHG]). Scale bar, 20 µm. (F) Whole mount images of the meninge in Tomato⁻ BM transplanted chimeric mice (red, Tomato⁺ macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, skull imaged by SHG). Note Tomato⁺ perivascular macrophages underneath skull. Scale bar, 20 µm. (G) Whole mount images of pancreas from Tomato⁻ BM transplanted chimeric mice (red, Tomato⁺ macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, collagen bundles shown as SHG) Scale bar, 50 µm. (H) Whole-mount images of the parietal peritoneal membrane from Tomato⁻ BM transplanted chimeric mice (red, Tomato⁺ macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, collagens shown by SHG). Scale bar, 50 µm, (E, G, and H) Note Tomato⁺ membrane-associated macrophages in collagen-enriched serosa membrane. (I) Comparison of liver capsular macrophages between Tomato⁻ BM-transplanted chimeric mice (left) and CX₃CR1^gfp/+ mice (right); red, Tomato⁺ macrophages; green, CX₃CR1gfp⁺ macrophages; blue, collagen. Scale bar, 50 µm. All two-photon microscopic images are representative of at least two independent experiments.