Rm2155 hard tissue microtome

Calcein (10 mg kg⁻¹) and xylenol orange (90 mg kg⁻¹) (both from Sigma‐Aldrich, St. Louis, MO, USA) were injected subcutaneously on days 13 and 3 before sacrifice, respectively. After termination, femurs were fixed in 4% paraformaldehyde solution, followed by dehydration using gradient ethanol, vitrification by xylene and embedded in methyl methacrylate. The bones were cut coronally into 10 µm thick sections with the RM2155 hard tissue microtome (Leica, Wetzlar, Germany). The double labeling images of bone slices were captured using a fluorescent microscope (Leica DM5500 system, Germany). Cortical bone formation was analyzed in four randomly selected visual fields in the midshaft of femur. Mineral apposition rate (MAR) was analyzed by OsteoMeasure Image Analysis System (Osteometrics, Decatur, GA, USA).

The femurs were 10% buffered formalin fixed, decalcified by 10% EDTA at 37 °C for 2 weeks and then embedded in paraffin. The paraffin-embedded tissues were sectioned at 7-μm thickness and performed with TRAP staining (Sigma-Aldrich, St Louis, MO, USA), Toluidine blue O staining (Sigma-Aldrich, St Louis, MO, USA) and IHC staining according to manufacturer’s instruction. For IHC staining, the sections were incubated with primary antibodies including rabbit anti-OCN (1:100, ab93876, Abcam, Cambridge, MA, USA) and rabbit anti-OPN (1:200, ab8448, Abcam, Cambridge, MA, USA) overnight at 4 °C. The horseradish peroxidase-streptavidin system (Dako, Carpinteria, CA, USA) was used for signal detection which followed by counterstaining with hematoxylin.
To process the un-decalcified femur specimens, the femurs were fixed by 10% buffered formalin overnight and embedded in methyl methacrylate. The samples were sectioned at 7-μm thickness by RM2155 hard tissue microtome (Leica, Wetzlar, Germany). The sections were stained by the von Kossa/nuclear fast red method as well as Masson-Goldner trichrome staining technique following the previous protocol [34 (link)]. The unstained 7-μm sections were used for dynamic histomorphometric analysis by a semi-automatic digitizing image analysis system (OsteoMetrics, Atlanta, GA, USA) as mentioned previously [35 (link)].