1% Trixon X 100. After blocking with 1% BSA in PBS, the cells were first incu bated with an anti vinculin mAb and then a tetramethylrhodamine isothiocya nate conjugated anti Crizotinib cost phalloidin rabbit polyclonal antibody. The former antibody was visualized using a fluorescein isothiocyanate conjugated secondary antibody. After staining, cells Inhibitors,Modulators,Libraries were observed Inhibitors,Modulators,Libraries under a fluorescence microscope. Evaluation of sulfated proteoglycan synthesis Quantitative assessment of proteoglycan synthesis in pellet cultured chondrocytes was performed by a previ ously described method. In brief, the culture medium was replaced with a fresh one containing 0. 1% fetal bovine serum and 10 uCi ml sulfate. After 4 hours of labeling, a pellet was recovered, rinsed extensively with ice cold PBS, and subjected to papain digestion at 55 C for 16 to 24 hours with gentle agitation.
The digest was centrifuged and the radioactivity of the supernatant was measured. The radioactivity was normalized by the DNA content of the supernatant, which was determined Inhibitors,Modulators,Libraries by the Quant iT dsDNA Assay Kit. Inhibitors,Modulators,Libraries Histological evaluations For histological evaluations, chondrocyte pellets were fixed in paraformaldehyde, embedded in paraffin, and sections 6 um thick were prepared. The sections were stained with hematoxylin and eosin, or Safranin O and fast green, and were observed under a light microscope. For immunohistochemistry, the sections were digested with 1. 0% hyaluronidase for antigen retrieval, and then incubated overnight with an anti type I collagen polyclonal antibody prepared at the concentration of 2 ug ml in PBS.
The antibody was finally visualized with the avidine Inhibitors,Modulators,Libraries linked peroxidase system coupled with 3 amino 9 ethylcarbazole substrate. Statistics Data were analyzed by paired t test or repeated measures one way factorial analysis of variance. If the analysis of variance showed significance, data were further analyzed by Fishers protec ted least significant difference test as a new post post hoc test. The level of significance was set at P 0. 05. First, the expression of type I and type III procollagen was evaluated sequentially for 1 week in primary cultured human articular chondrocytes maintained in monolayers. In those cells, the expression of both procollagen genes increased dramatically after plating, confirming the results of previous studies. Of these two genes, the increase was more obvious with type I procollagen, which showed a nearly eightfold increase in the first 7 days after plating. In the following part of this study, we attempted to clarify the mechanism for this induction of noncartilaginous procollagen gene expression. Previously, we determined 11 dominant integrins in human articular chondrocytes.