Anti ocn

Total protein was extracted using RIPA lysis buffer with protease and phosphatase inhibitors (Solarbio, China) at 4 °C. Protein concentration was determined using a BCA Protein Assay Kit (EpiZyme, China). Equal amounts of protein (30 μg) were subjected to 10% (w/v) SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). After blocking with 5% (w/v) BSA, the membrane was incubated with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000; 115-035-003, 111-035-003, Jackson ImmunoResearch, USA) at 25 °C for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Millipore, USA) and the grayscale of protein bands were semi-quantified using ImageJ software.
The primary antibodies used in this study included anti-PPARγ (1:1500; #2435, Cell Signaling Technology, USA), anti-IκBα (1:1500; #4814, Cell Signaling Technology, USA), anti-p65 (1:1500; #8242, Cell Signaling Technology, USA), anti-phosphorylated p65 (p-p65; 1:1500; #3033, Cell Signaling Technology, USA), anti-BMP-2 (1:1500; ab214821, Abcam, UK), anti-OSX (1:1500; ab209484, Abcam, UK), anti-OCN (1:1000; A6205, ABclonal, China), and anti-GAPDH (1:2000; #5174, Cell Signaling Technology, USA).

For histological analyses, after 2 (n = 3) and 4 (n = 3) weeks of consolidation, the tibia specimens were fixed in 4% (w/v) PFA for 24 h, decalcified in 10% (w/v) EDTA for 21 days, dehydrated through graded ethanol of increasing concentration, and then embedded in paraffin. Samples were cut into 5-μm-thick longitudinally oriented sections and processed for hematoxylin-eosin (H&E), Masson's trichrome, and Safranine O-Fast Green (SO-FG) staining.
For immunohistochemical staining, sections were incubated in 0.3% (v/v) hydrogen peroxide for 20 min to quench endogenous peroxidase activity. After antigen retrieval in 0.01 mol/L citrate buffer (pH 6.0) at 65 °C for 20 min and blocking with 5% (v/v) goat serum for 1 h, sections were incubated with primary antibodies at 4 °C overnight. After incubation with secondary antibodies (1:1000; 111-035-003, Jackson ImmunoResearch) conjugated with HRP at room temperature for 1 h, an HRP-streptavidin system (Dako, Denmark) was used to detect positive areas followed by counterstaining with hematoxylin. Primary antibodies used in this study included anti-OCN (1:100; A6205, ABclonal, China) and anti-β-catenin (1:100; #8480, Cell Signaling Technology).