Evaluating Osteoarthritis Progression in Rat Knees

Animals were sacrificed three weeks after lubricin injection (four weeks after DMM surgery) and intact knee joints were dissected and fixed in 4% normal buffered formalin (Sigma, St. Louis, MO). Samples were decalcified and embedded in paraffin (VWR, Radnor, PA). Ten μm thick, longitudinal serial sections were stained with Safranin O (Fisher, Waltham, MA) to visualize proteoglycans. Whole joint sections, medial, lateral and the ACL/PCL insertion sites were graded for signs of OA according to the OARSI Guidelines for rat knee joints34 (link). Sections were deparaffinized in CitraSolv (Fisher Scientific; Fairlawn, NJ) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0, Fisher Scientific) and blocking (1:500 dilution; 100 μL rat serum:50 mL TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 hr), steps were performed prior to going through sequential wash (TBST) and primary antibody application steps. A primary antibody for NFkB P65 antibody (Cell Signaling Technology), or lubricin12 (link) bound to Alexa 488 (Molecular Probes) and the nucleic acid stain DAPI (Sigma) were applied to sections. After antibody staining, sections were mounted using FluorSave reagent (Calbiochem) and coverslipped.