Immunostaining for Cartilage Markers

The expression levels of type II collagen (COLII) and aggrecan (ACAN) were assessed in a cell culture using specific antibodies obtained from Sigma-Aldrich, Madrid, Spain, as previously documented [31 (link)]. Scaffolds containing cells were transferred onto 8-well cell culture slides (Millicel, Sigma-Aldrich, Madrid, Spain). The cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min, then washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Following three washes, they were incubated for 30 min with a blocking solution (1% bovine serum albumin [BSA] and 1.1% Tween-20 in PBS). Subsequently, the scaffolds were incubated overnight at 4 °C with the appropriate primary antibodies (diluted in antibody diluent solution at 1:100 for ACAN and 1:500 for COLII antibody). After three additional washes, the scaffolds were incubated with the following corresponding secondary antibodies: an anti-mouse (for COLI and COLII) or anti-rabbit (for ACAN) FITC-conjugated antibody (Sigma-Aldrich, Madrid, Spain), diluted at 1:200. Following the final washes, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the samples were examined using a Leica DM2500 fluorescence microscope (Leica, Wetzlar, Germany)