Fig. 7. Evidence for osteogenic and chondrogenic differentiation in neural crest cultures grown for 28 days. (A) Culture stained with both Alcian Blue (c) and von Kossa (asterisk), indicating the existence of cartilage matrix and calcium phosphate mineralisation respectively. (B) Transverse section through a von Kossa-stained nodule containing hypertrophic chondrocytes (arrows) and areas of new mineralisation (arrowheads). (C,D) Confocal images of cultures stained immunohistochemically for osteonectin. Osteonectin is expressed (C) in the extracellular matrix and (D) intracellularly. (E) Transmission electron microscopy showing ultrastructure of a chondrocyte undergoing hypertrophy, a pale cell with swollen endoplasmic reticulum cisternae (arrowheads). (F) Osteoid matrix formation in association with mineralised areas of nodule. Mineralised matrix stains with von Kossa, and is bounded by regions of hypertrophying cartilage (h) and osteoid matrix (arrowheads). (G,H) Bone formation in 28 day cultures. (G) A sheet of alizarin red positive cells (b) is lying away from Alcian Blue-positive cartilage nodules (c) suggesting endochondral bone formation. (H) An Alizarin Red-positive area (arrowheads) within an Alcian Blue-stained region of cartilage showing endochondral bone formation. Scale bars: 10 µm in A,C,D; 50 µm in B,F; 5 µm in E; 1 mm in G; 0.1 mm in H.