Alcian blue staining

The cells (passage 0) used in this analysis were fixed in 3.5% paraformaldehyde in PBS for 15 min, and then stained with 0.1% Alcian blue in 0.1 M HCl overnight. The chondrocytes were washed three times with PBS buffer and 6 M guanidine HCl was added for 6 h. The production of sulfated proteoglycan was measured at 595 nm. Rabbit joint cartilage explants were fixed in 4% paraformaldehyde in PBS for 24 h at 4℃, washed with PBS, dehydrated with graded ethanol, embedded in paraffin, and sectioned at 4 mm thickness.²² Cartilage sections were deparaffinized in xylene and sequentially rehydrated in graded alcohol samples. After washing with PBS, cartilage sections were stained with 2.5% Alcian blue solution followed by three washes with 0.1 N HCl. After the sections were rinsed with PBS, the nuclei were counterstained with hematoxylin, and each section was observed under a light microscope (100× magnification). The data represent results of a typical experiment from at least four independent experiments.