Anti sclerostin

Paraffin sections were used for immunohistochemical analysis. Briefly, sections were deparaffinized, treated with 3% H2O2 to inhibit endogenous peroxidase activity, blocked with rabbit or goat serum, and then incubated with anti-Hif-1α (Abcam,USA), anti-Hif-2α (Novus,USA), anti-Sclerostin (Santa Cruz,USA), anti-Dmp-1 (Biovision,CA) and β-catenin (Abcam, USA), at 4°C Overnight. Followed by biotinylated secondary antibodies and a peroxidase-labeled streptavidin–biotin staining technique (DAB kit, Invitrogen), nuclei were counterstained with hemalum (FARCO Chemical Supplies, Hong Kong). The slides were visualized by a microscope (ZEISS, AXIO). The slides without incubation with secondary antibody were used as negative controls.

The phosphorylation of ERK1/2 and JNK, the activation of caspase‐3 and the expression of RANKL, OPG, sclerostin, GSTP1‐1, and MKP‐1 were performed by western blot in MLO‐4Y treated as above reported. Cells were lysed in ice cold RIPA buffer containing phosphatase and protease inhibitor cocktails (Sigma) and centrifuged at 11 600 g for 10 min. Equal amounts of total proteins (40–60 μg) from whole‐cell extract were subjected under reducing conditions to SDS/PAGE on 10% gel and electrotransferred to PVDF membrane (GE Healthcare). Proteins were visualized by incubating the membranes with specific primary antibodies: anti‐caspase 3 or anti‐phospho‐ERK 1/2 or anti‐phospho‐JNK (Cell Signalling Technology, Beverly, MA, USA), or anti‐RANKL or anti‐OPG or anti‐sclerostin or anti‐GSTP1‐1 or anti‐MKP‐1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, membranes were stripped and reprobed with anti‐ERK1/2 or anti‐JNK or anti‐β‐actin for normalization and densitometric analysis. Secondary antibodies conjugated to horseradish peroxidase were used to detect antigen–antibody complexes with a chemiluminescence reagent kit (Bio‐Rad, Hercules, CA, USA). image j software (National Institutes of Health, Bethesda, MD, USA) was used to perform quantitative analyses, and band values were expressed as percentage relative to values of control.