AB H E or Wortmannin SO FG stained areas were outlined on projected images of each histologic section to determine articular cartilage area and thickness. Immunohistochemistry and TUNEL staining Three um thick paraffin sections were baked at 60 C over night. Slides were then deparaffinized, rehydrated, and digested with pepsin. DAKO endogenous blocking reagent was then used to quench endogenous peroxidase for 10 minutes. Normal horse serum or normal goat serum was used to block non specific binding sites for 20 minutes. Collagen II or collagen X primary antibodies were added and the slides were incubated at 4 C overnight. Secondary biotinylated horse anti mouse antibody or goat anti mouse antibody was added for 30 minutes on the second day, followed by incubation with streptavidin for 30 minutes.
Positive staining was detected by Romulin AEC Chromagen. Inhibitors,Modulators,Libraries To detect chondrocyte apoptosis after meniscal surgery, terminal deoxynucleotidyl trans ferase dUTP nick end labeling Inhibitors,Modulators,Libraries staining was performed using a kit based on the manufacturers instruc Inhibitors,Modulators,Libraries tion. Grading of cartilage structure Tissue sections were stained with Alcian blue Orange G and graded by two blinded observers based on the scoring system developed by Chambers et al. In brief, each section was assigned a grade as follows, 0 normal carti lage, that is, lack of superficial zone fibrillation or clefting, 1 mild superficial fibrillation, 2 fibrillation and or clefting extending below the superficial zone, 3 mild loss of articular cartilage, 4 moderate loss of articular cartilage, 5 focal loss of cartilage to the subchondral bone, 6 severe loss of articular cartilage.
The progression of OA in differ ent groups was evaluated using this method. Three slides Inhibitors,Modulators,Libraries of each sample were analyzed and five mice were used in each group. MMP13 activity assay The ability of CL82198 to inhibit MMP13 activity in vitro was determined using the SensoLyte 520 MMP13 Assay Kit. Five ng of MMP13 and 10 ug mL of Inhibitors,Modulators,Libraries CL82198, or control substrate were added into the MMP13 Assay Kit. MMP13 activity was detected following the manufacturers instructions. Primary sternal chondrocytes were isolated from three day old wild type pups as previously described. Cells were plated in 12 well plates and treated with bone morphogenetic protein 2 alone or BMP 2 with the MMP 13 inhibitor CL82198 for 60 hours.
The culture media were collected and MMP13 activity was determined using the SensoLyte 520 MMP13 Assay Kit following the manufacturers instructions. Statistical analysis Results of all quantitative assays involving multiple doses selleck chem inhibitor and time points were analyzed using analysis of variance followed by Dunnetts test. For experiments comparing two groups, unpaired students t test was applied. P 0. 05 was considered to be a significant difference.