Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S.
A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.
D000818:Animals; D001718:Birefringence; D001842:Bone and Bones / Q000473:pathology / Q000648:ultrastructure*; D002118:Calcium / Q000032:analysis*; D002356:Cartilage / Q000648:ultrastructure*; D002642:Chick Embryo; D006651:Histocytochemistry; D006984:Hypertrophy / Q000473:pathology; D013194:Staining and Labeling
D002118:Calcium