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-(�9-amino-ethyl ether)N-N’-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pencellular 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% sucrose at pH 4.0-4.5 for optimal staining. The stained tissue blocks were rinsed and 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-bounded 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. In 1971 (5) a labile form of calcium was reported to be present in the cytoplasm of epiphyseal chondrocytes using the glyoxal bis(2hydroxyanil) (GBHA) method. The cellular calcium was localized as 0.5-s granules distributed throughout the cytoplasm. Attempts to identify the organelles containing the Ca-GBHA granules using electron microscope failed because the Ca-GBHA complex was uncoupled in the various plastic-embedding media tested. Furthermore, cytomembranes were destroyed by the alkaline, alcoholic solvent necessary to dissolve the GBHA powder. To localize this labile form of intracellular calcium for electron microscopy, therefore, it was necessary that a calcium-binding compound be found which is soluble in water, precipitates calcium and retains the cytomembranes. Prompted by these specifications, a variety of water-soluble compounds were tested