Alcian Blue Staining to Visualize Proteoglycans: A Technique to Detect Proteoglycans in Mesenchymal Stem Cell Differentiated Chondrocytes Culture In Vitro

To carry out chondrogenic differentiation from a sponge-shaped medical device constituted from lyophilized collagen type 1, cut cubes 3 millimeters per side. Seed MSCs on top of the cubes at a concentration of 4 x 10⁶ cells/ml.

After allowing the cells to adhere to the cubes for 30 minutes at room temperature, maintain MSC-collagen constructs in chondrogenic medium, as listed in the text protocol. Add 0.4% Alcian blue solution to the sections, and incubate overnight at 4 degrees Celsius.

The next day, use 40% DMSO and 0.05 molar magnesium chloride to wash the cells for 30 minutes before using nuclear fast red to counterstain the cells.