Sclerostin

Western blotting of whole-cell extracts isolated from cells in culture after FSS or extracts isolated from murine long bone was done as previously described (68 (link), 70 (link)). Equal amounts of protein were loaded and electrophoresed on 10% SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% nonfat dry milk (unless otherwise stated) and probed with the indicated primary antibodies overnight at 4°C. Antibodies were detected with the appropriate horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology) and enhanced chemiluminescence detection reagent (Bio-Rad). The antibodies used were sclerostin (R&D Systems, AF1589), α-tubulin (Sigma, T9026), detyrosinated tubulin (Abcam, ab48389), phospho-CamKII Thr²⁸⁶ (Cell Signaling Technology, 12716S), total CaMKII (Cell Signaling Technology, 11945S), and GAPDH (Millipore, MAB374). Blots were acquired using an EpiChem gel documentation system (UVP Bioimaging Systems) and analyzed using ImageJ software.

We harvested fractured tibiae at 7, 10, and 14 days. Subsequently, we fixed the tissues in 10% formalin for 24 hr and decalcified in 10% EDTA at pH 7.2 for 2 weeks. The samples were processed, embedded in paraffin, and cut into 3-μm sagittal sections. Then, we stained three contiguous sections (100 μm apart) from each specimen with Alcian Blue/Hematoxylin/Orange G (ABH) according to standard protocols.
We conducted quantitative analysis on the sagittal ABH/OG-stained sections by histomorphometric analysis using Osteometrics software. Parameters included the areas of woven bone, cartilage, and total fracture callus, and calculated the amount of mesenchymal tissue by subtracting bone and cartilage area from the total callus area. We expressed bone, cartilage, and mesenchymal tissue areas each as a percentage of the total fracture calluses. Sections were stained with a polyclonal anti-mouse antibody specific for β-catenin (1:50, Cell Signaling Technologies, Danvers, MA) or sclerostin (1:100; R&D Systems), or matched isotype control antibody. The primary antibody was visualized with an HRP-conjugated rabbit anti-goat antibody (Jackson ImmunoResearch Laboratories). Sections were counterstained with hematoxylin.