Undecalcified Bone Histological Analysis

The animals were sacrificed by cervical dislocation, and the left tibia was dissected. Cortical bone sections were prepared and stained using a protocol for undecalcified bone [54 (link)]. Briefly, immediately after dissection, the bones were fixed in Karnovsky’s fixative and cut with a diamond blade saw (Buehler, Co., Dusseldorf, Germany) in the transverse plane at 50% of the bone biomechanical length. The cortical sections were ground to a final thickness of ∼ 50 μm using Carbimet paper discs (800 and 1200 grit; Buehler, Co., Dusseldorf, Germany), dehydrated in ascending graded ethanol and stained with fluorescein isothiocyanate (FITC; Sigma-Aldrich, Co., St. Louis, MO, USA).