Pronase p 8811

Cartilage sections were dewaxed in decreasing graded ethanol solutions until PBS rinsing for 20 min and then immunostained for TGFβ1 and Coll II. Briefly, after fixation, sections were extensively rinsed in PBS and permeabilized by incubation in 0.3 % hydrogen peroxide in PBS solution for 15 min. For Coll II immunostaining, sections were pre-treated for antigen unmasking with 0.2 % Pronase (P-8811, Sigma, Mo, USA) solution in PBS for 30 min at 37 °C. Then, 10 % normal serum was added for 1 h at room temperature to block nonspecific antibody binding and the primary antibodies (rabbit polyclonal antibody anti TGFβ1, sc-146 and mouse monoclonal antibody anti collagen II, sc-52658, 1:50 dilutions, Santa Cruz Biotechnology, CA, USA) were applied and incubated overnight at 4 °C. After rinsing in PBS, slides were incubated with appropriate biotinylated secondary antibody and with horseradish peroxidase-streptavidin complex for 1 h each (ABC Staining System, Biotechnology, CA, USA). Sample reaction was developed with 3,3-diaminobenzidine substrate and permanently mounted. Negative controls, by omitting the primary antibody, were included to check proper specificity and performance of the applied reagents.