Extended Data Fig. 4: Analysis of MC network formation and periarteriolar alignment in vivo.

(a) Homogenous MC distribution and periarteriolar MC alignment in the dermis of IgE-deficient Rag2^−/− mice. Representative ear skin whole mount immunofluorescence staining of an 8-week old adult mouse of n = 3 mice: collagen IV (white), avidin (cyan), ACTA2 (red). (b) Histological quantification of MC density in the dermis of ear skin whole mount tissue. Each dot represents the average value of four imaging field of views from one mouse (n = 11 mice per genotype); P = 0.6026, ns, non-significant, two-sided t test. Bars display the mean. (c,d) Two experimental strategies to identify dermal vessel types were applied for the analysis of MC proximity to different blood vessels. (c) Workflow for the post-imaging analysis of MC proximity to arterioles, venules and capillaries with Imaris software. This analysis was based on immunofluorescence stainings of ear skin whole mount for MCs (avidin), α-smooth muscle actin (ACTA2) and endomucin (EMCN). ACTA2⁺/EMCN⁻ vessels were classified as arterioles, ACTA2⁺/EMCN⁺ vessels as venules and ACTA2⁻/EMCN⁺ vessels as capillaries. This workflow was used for the data analysis in Fig. 2c. (d) Workflow for the post-imaging analysis of MC proximity to arterioles, venules and capillaries with Imaris software. This analysis was based on immunofluorescence stainings of ear skin whole mount for MCs (avidin), α-smooth muscle actin (ACTA2) and ACKR1. ACTA2⁺/ACKR1⁻ vessels were classified as arterioles and ACTA2⁺/ACKR1⁺ vessels as postcapillary venules (see Fig. 2b). MC proximity analysis was comparable to the results obtained with ACTA2/EMCN stainings in Fig. 2c. Dots represent individual imaging fields of view (n = 12, 12), which come from five adult mice; *** P < 0.0001 (Art.), P = 0.4370, ns (Ven.); two-sided t tests. Bars display the mean. Scale bars: 300 µm (a), 100 µm (c,d).

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