Visualizing Neocartilage Deposition and Distribution

To visualize neocartilage deposition and distribution, immunofluorescence of collagen I, II, and X and histological staining of sGAG were performed on encapsulated chondrocytes after 3 weeks in culture. Cell-laden hydrogels were first fixed with 4% paraformaldehyde and dehydrated using 30% sucrose solution overnight before freezing in OCT compound (Tissue-Tek). Sections were prepared and stained for type I, II, and X collagen using rabbit polyclonal antibodies (Abcam) in accordance with a previously reported immunostaining protocol.^{24 (link)} For sGAG histological staining, sections were prepared and stained as previously reported^{25 (link)} using hematoxylin solution (Sigma-Aldrich) for nuclei staining and 0.1% safranin-O solution (Sigma) for staining sGAG.