Stp6000

C57/BL6N mice were intraperitoneally injected with 20 mg/Kg calcein (Sigma-Aldrich, USA) dissolved in PBS at a concentration of 2 mg/ml with 1 mg/ml NaHCO3 (Sigma-Aldrich, USA) at 7 d and 2 d before killing. After killing, the femurs were obtained, fixed in 4% formaldehyde, and embedded in methyl methacrylate. A hard tissue slicing machine (SP1600; Leica, Germany) was used to sagittally section specimens into 30-mm sections away from light. Then, the cortical endosteum surfaces were evaluated using a fluorescence microscope (STP6000; Leica, Germany) with an excitation wavelength of 488 nm. The bone formation rate (BFR), mineral deposition rate (MAR), and recommended relevant calculations were used for quantitative analysis using ImageJ software.

Cells were fixed with 4% paraformaldehyde for 20 min, blocked with 1% BSA in PBS for 1 h, and incubated with 1:100-diluted primary antibodies for 4 h at 4 °C. Next, the cells were washed with PBS and incubated with 1:200-diluted Alexa Fluor 594-conjugated second antibodies for 1 h at 4 °C. Finally, the cells were mounted in glycerol containing 4’-6-diamidino-2-phenylindole (DAPI), and examined using a fluorescence microscope (Leica STP6000).

calcein labelling was applied to examine bone formation rates, as stated before^{9 (link),25 (link)}. Mice received intraperitoneal injections of calcein (20 mg/kg, Sigma-Aldrich, USA) at 16 d and 2 d prior to sacrifice. At sacrifice, the right femora were isolated, fixed in 80% ethanol, and embedded in methyl methacrylate. The specimens were sagittally cut into 30-μm thick sections and both double-labelled and single-labelled cortical endosteum was detected using a fluorescence microscope (STP6000; Leica, Germany) with an excitation wavelength of 488 nm. Quantification was determined using the ImageJ 1.47 software to evaluate mineral apposition rate (MAR) and bone formation rate (BFR)^{9 (link),25 (link)}.
TRAP staining was applied to examine bone resorption rates, according to previous studies^{9 (link),25 (link)}. After being isolated, tibiae were fixed with paraformaldehyde, decalcified with 10% ethylene diamine tetraacetic acid (pH, 7.2-7.4), and embedded in paraffin. The proximal metaphyses were sagittally sectioned into 5-μm sections using a microtome (RM2125; Leica, Germany). The sections were stained with TRAP by a commercial kit according to the manufactures’ instructions (387-1 A; Sigma-Aldrich, USA). Parameters of number of osteoclasts per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS) were quantified using the ImageJ 1.47 software^{9 (link),25 (link)}.

Balb/c female mice were intraperitoneally injected with 20 mg/kg calcein (Sigma–Aldrich, USA), dissolved in PBS at a concentration of 2 mg/ml with 1 mg/ml NaHCO3 (Sigma–Aldrich, USA) at 16 d and 2 d before sacrifice. After sacrifice, the left femur was obtained, fixed in 4% formaldehyde, and embedded in methyl methacrylate. The specimen was cut into 30 mm slices by using a microtome (SP1600; Leica, Germany) under the condition of avoiding light. Then, cortical endosteum surfaces were evaluated using a fluorescence microscope (STP6000; Leica, Germany) with an excitation wavelength of 488 nm. Bone formation rate/bone surface area (BFR/BS), osteoblast surface area/osteoblast surface area (Ob.S/BS), osteoclast surface /bone surface area (Oc. S/BS) and number of osteoclast per bone surface (N.Oc/B.Pm) were used for quantitative analysis using ImageJ 1.47 software.

Imaging was performed using Leica STP6000 and Leica TCS SP5 inverted confocal microscope. The images were captured and analyzed with the LAS AF software.