Bone development and mineralization in F/I KO mice. (a) Cartilage matrix (blue) and mineralization (red) are showed by Alcian blue + alizarin red staining. (A) Whole mount staining of F/I KO mice (left) and control mice (right). Mineralization is decreased/delayed in the areas where Alcian blue staining is stronger in the KO mice. (B) The limb of F/I KO mice (above) and control mice (bottom). Mineralization is significantly decreased in the humerus and fingers (arrow). (C) Feet of F/I KO mice (left) and control mice (right). The toe bone of F/I KO mice are less mature than the control (arrow). (D) Lumbar of F/I KO mice (left) and control mice (right). Alcian blue staining showed more cartilage matrix in the KO group (arrow). (b) Bone histology and histomorphometry. (A) H&E staining of longitudinal sections of the femur (upper; ×4 magnification; scale bar = 200 μm). H&E (middle) and TRAP staining (lower) of primary ossification centers (×20 magnification; scale bar = 50 μm). (B) Histomorphometric quantification of Tb.Ar%, Ob.N/B.Pm, and Oc.N/B.Pm. (c) Representative three‐dimensional model reconstructions of live animal μCT scan data shows the mineralization of the skull (first row), hyoid (second row), ribs and xiphoid (third row) and coccyx (last row) of the F/I KO mice and control mice at 6, 10, and 18 weeks. (d) Cort. BMD (A), Cort. BS/BV (B), Trab. BV/TV (C), Tb.Sp (D), Tb.N (E), and Tb.Th (F) in femurs of knockout and control mice at 6, 10 and 18 weeks (n = 5). Values are shown as means ± SD. CON, control; Cort.BMD, cortical bone mineral density; Cort.BS/BV, cortical bone surface/bone volume; F/I KO, Osx‐Cre:FNDC5/Irisin KO mice; H&E, hemotoxylin and eosin; KO, knockout; Ob.N/B.Pm, osteoblast number per bone perimeter; Oc.N/B.Pm, osteoclast number per bone perimeter; Tb.Ar%, trabecular bone area; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; Trab.BV/TV, trabecular bone volume/tissue volume; Trap, tartrate‐resistant acid phosphatase; μCT, microcomputed tomography. *p < .05 versus control