Histology and Bone Histomorphometry.

Decalcified and nondecalcified sections of bone were obtained, as described previously (65). Briefly, mice were injected s.c. with calcein (10 mg/kg; Sigma) 8 d (adult mice) or 12 d (aging mice), after which they received a Xylenol orange (90 mg/kg; Sigma) injection. Lumbar vertebrae (L1–4) were fixed with 4% (vol/vol) paraformaldehyde for 18 h at 4 °C. Nondecalcified bones were embedded in optimum cutting temperature compound (O.C.T. compound, Tissue-Tek), and 5- to 7-µm-thick frozen sections were prepared using a transparent film. Static and dynamic histomorphometric analyses were performed according to standard protocols using software kindly provided by Robert J. van’t Hof.