Fig. 5. Analysis of growth plate extracellular matrix (ECM) markers of the proximal tibia at E18.5. (A) Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis of ECM glycosaminoglycans (GAGs). (B) Chondroitin sulfate (CS) immunohistochemistry showing ECM staining (black arrow) in all three growth plate layers (p, proliferating chondrocytes; c, columnar chondrocytes; h, hypertrophic chondrocytes) of wild-type cartilage (+/+), whereas CS staining in mutant (gt/gt) proliferating and columnar layers is restricted to the pericellular space (arrowhead), with very little staining in the ECM (black arrow). However, in the columnar and hypertrophic layers of mutants, ECM staining of CS was observed (white arrows). (C) C6S was detected by immunofluorescence staining and is distributed in the outer-most layers of the proliferative zone in wild-type cartilage (+/+), whereas in mutant cartilage (gt/gt), low pericellular C6S staining was observed in both proliferative and columnar layers. (D) Distribution of aggrecan was analyzed by immunofluorescence, which revealed localization to the ECM in all layers of the wild-type cartilage (+/+), as well as in the proliferative layer of mutant cartilage (gt/gt). However, in the mutant, aggrecan was increasingly restricted to the pericellular space in columnar and hypertrophic (arrowhead) layers. (E) Detection of collagen II by immunofluorescence shows strong staining of the ECM in both wild-type (+/+) and mutant (gt/gt) proliferative and columnar layers, and decreased staining in the ECM of the hypertrophic layer. Scale bar: 10 µm.