Dako envision system hrp labelled polymer detection kit

Immunostaining was performed as previously described.31 (link) Briefly, 4 µm sections (intervening levels between the sections used for pathology scoring) from 5 to 8 randomly selected mice from each group, were dewaxed, rehydrated and digested with 500 U/mL bovine testicular hyaluronidase (Sigma-Aldrich, St. Louis, MO) before overnight incubation with polyclonal rabbit anti-rat matrix metalloproteinase (MMP)-13 that cross reacts with mouse32 (link) (LSBio, LS-B3168, 1.5 µg/mL), in Dako antibody diluent (#S0809, Agilent Technologies) or isotype-matched IgG. For immunodetection, Dako EnVision+System HRP labelled polymer detection kit (Dako) was used with ImmPACT NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, California, USA), counterstained by Mayer’s haematoxylin and Scott’s bluing solution, and digital images acquired (Nano Zoomer). All samples were stained simultaneously to exclude between-run variability. The number of MMP-13 positive chondrocytes in anterior, central and posterior regions of tibial and femoral non-calcified and calcified cartilage (demarcated by tidemark) were counted (average from two independent scorers), summed and presented as either total tibial, femoral or tibiofemoral. The percentage of a standard ROI in the anterior tibial synovial fossa stained for MMP-13 was quantified using ImageJ.