Pten^−/− mice have circulating MCs and increased MC numbers in tissues. (A) Tissue samples of the indicated tissues from 8-week-old mice were fixed with 4% formalin, paraffin-embedded, and sections were stained with toluidine blue. MCs from Pten^−/− mice showed spindle-shaped and partially degranulated phenotypes (inset). MC numbers per millimeter squared (graphs) were determined by counting blinded samples from individual mice. Images were visualized on a Leica DM LB2 microscope using a 20× (0.40 NA) objective for dry mounted slides. Image capture was with a QImacong Micro Publisher 3.3 RTV camera using QCapture 2.7.3 software. Image analysis was with ImageJ 1.45 (National Institutes of Health). Statistical analysis was by an unpaired 2-tailed Student t test: *P < .05; **P < .01. Scale bar represents 100 μm. (B-C) Percentage of FcϵRI and Kit-positive cells was analyzed in peripheral blood (B) and in the bone marrow (C) of individual mice with the indicated genotype. Cells (1 × 10⁵) were labeled with PE-MAR-1 (anti-FcϵRI) and APC–anti-Kit. Mean fluorescence intensity was measured by flow cytometry (n = 15 for peripheral blood cells; 6 of 15 mice had circulating MCs; n = 11 for bone marrow cells). For peripheral blood cell counts, 0.025% of double = positive cells was set as a threshold for background. (B-C) A minimum of 4 individual experiments were conducted.