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

Proteoglycans are composed of a core protein covalently attached to sulfated glycosaminoglycan chains.

To visualize proteoglycans, begin with collagen-based scaffolds - the porous polymeric substrate - and seed the mesenchymal stem cell, or MSC, suspension onto them. This provides structural support to the MSCs and allows them to grow on the surface as well as inside the cavities of the scaffolds.

Treat the constructs in a chondrogenic differentiation medium containing transforming growth factors that drive the transformation of MSCs into mature chondrocytes - cartilaginous cells. Mature chondrocytes secrete proteoglycans that form a network with the collagen in the scaffold.

Take a section prepared from the chondrocyte-laden construct. Add an acidic solution of alcian blue stain supplemented with guanidium hydrochloride and incubate. During incubation, the guanidine hydrochloride in the dye solution dissociates any proteoglycan aggregates for better staining.

Subsequently, the negatively charged sulfated glycosaminoglycans from the proteoglycans bind to the copper-containing positively charged alcian blue dye molecules, resulting in blue deposits.

Next, immerse the slide in a destaining solution containing magnesium chloride. The magnesium ions compete with the positively charged dye molecules for binding sites on the glycosaminoglycans, thus, preventing overstaining.

Image the section. Blue coloration confirms the presence of proteoglycans in the sample