The percentage of osteocytes that express detectable levels of sclerostin-IR increases with age. (A) Low-power magnification, medial-lateral view H&E section of a young mouse femur showing the regions where osteocyte sclerostin-IR was analyzed in young (4-month-old), middle-aged (9-month-old), and old (24-month-old) adult C3H male mice. Three sites were selected along the diaphysis: the proximal (4 mm from the proximal head), mid-diaphysis (8 mm from the proximal head), and distal (3 mm from the distal head). (B–D) High-power confocal images of sclerostin-IR (upper panel), DAPI (middle panel) and sclerostin/DAPI overlay (lower panel) in mouse femoral cortical bone of young (B) middle-aged (C) and old (D) adult animals. (E) Histogram illustrating the percentage of osteocytes expressing detectable levels of sclerostin-IR in cortical bone in young, middle-aged, and old adult diaphyseal cortical bone. All ages were statistically significantly different from each other at each site, except middle-aged vs. old at the proximal diaphyseal region (p>0.06). The numbers in parentheses above each bar indicate the number of animals analyzed for each age group. Bars represent the mean ± SEM; p<0.05 after a one-way ANOVA, Tukey’s post-hoc test. Scale (A) 1 mm; (B–D) 5 µm.