A simple pocedure for localizing calcium ions in tissues and in cells with glyoxal bis (2-hydroxy-anil) (GBHA) has been described. The GBHA molecule, in alcoholic solution made alkaline with NaOH, chelated with Ca, Ba, Sr, Cd, Cu, Co, and Ni ions, forming colored precipitates. All the colored GBHA complexes, with the exception of the red Ca-GBHA granules, were decolorized when immersed in a solution containing carbonate and cyanide. Of the methods available for demonstrating calcium ions, the sensitivity of the GBHA method was comparable only to the Scott-Packer electron microscope method; the latter, however, localized calcium plus magnesium while the GBHA method was specific for calcium ions. In tissues that were fixed in 10% neutral formalin, alcohol-formalin or absolute alcohol, false localization due to binding of extraneous or diffused calcium ions to polyanionic sites was evident. The false localization of calcium ions was prevented by processing the tissues by the freeze-dry or the freeze-substitution method, dehydrating the tissue below the cutectic point of calcium chloride. The GBHA method was not able to demonstrate calcium ions present as insoluble salts, such as calcium carbonate or as apatite crystals, giving false negative results.

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SYNOPSIS IN INTERLINGUA LE LOCALISATION DE CALCIUM IN CELLULAS ODONTOGENE DE DENTES MANDIBULAR DEL RATTO PER MEDIo DEL METHODO A GLYOXAL-BIS(2-HYDROXYANIL).-Blocos fresc de mandibula, de un spissitate de 1 mm, esseva obtenite ab rattos sugente e tincturate durante 16 horas in un solution de glyoxal-bis(2-hydroxyanil) (GBHA) e postea dishydratate in ethanol, acclarate in xyleno, includite in paraffin, sectionate a un spissitate de 7ju, e affixate per pression digital a albuminisate lamellas de vitro. Le lamellas esseva immergite in un solution de Na2CO3 e KCN pro assecurar specificitate pro calcium e contratincturate con blau methylenic. Granulos rubie de Ca-GBHA esseva localisate in le cytoplasma de adamantoblastos e cementoblastos e in le processes odontoblastic. Calcium in apatite non esseva tincturate.A method for localizing intracellular calcium using glyoxal bis(2-hydroxyanil) (GBHA) was recently developed in this laboratory.' By staining fresh osseous tissues with GBHA, calcium was demonstrable in cytoplasm and processes of osteoblasts, osteocytes, as well as in cytoplasm of periosteal cells and epiphyseal chondrocytes. Using the same GBHA staining procedure, an attempt was made in this investigation to localize calcium in cells of unfixed developing rat teeth.No reports were found that describe calcium in cytoplasm of ameloblasts, odontoblasts, and cementoblasts. However, Watt2 has proposed that calcium may be secreted by the odontogenic cells during tooth formation and by the osteogenic cells during bone formation. His secretary hypothesis was not based on actual observation of calcium in these cells, but merely deduced from his observations on the proximity of the early intercellular calcium deposits to cells.The present report shows calcium as red Ca-GBHA granules in cells that are responsible for formation of enamel, cementum, and dentin.

Fresh tibias and occipital bones from newborn mice and rats, as well as from 18-day-old mouse and chick embryos, were stained with 10% GBHA and 5% AgN03 solutions to localize calcium and phosphate or carbonates in spherules within the cytoplasm of chondrocytes and in the matrix of the calcifying cartilages. The mineralized spherules in the matrix adjacent to the hypertrophied chondrocytes were approximately 0.5 to 1 B in diameter. This was comparable in size to the spherules found within the cytoplasm of the chondrocytes. The spherules within the core of the spicules distal to the hypertrophied chondrocytes were about 2-3 P in diameter. The larger spherules were shown to contain an organic core which stained metachromatically with toluidine blue and was also stained with aldehyde fuchsin. The spherules were not visible after PAS reactions due to the intense staining. The labile GBHA and silver acetate positive spherules localized in the chondrocytes may be the source of the extracellular mineralized spherules. phosphate was perhaps complexed to an organic compound which was responsible for the spherical morphology. The present investigation was conducted to determine whether the cellular materials were deposited on the extracellular matrix as spherules to initiate calcification. This report was confined to studies on calcifying cartilages which precede endochondral ossification. MATERIALS AND METHODSCalcifying epiphyseal cartilages of tibia from newborn rats and mice were studied cytochemically to determine the sequence of mineralization. Occipital bones from 18-day-old mouse and chick embryos were also used to study an early stage in the sequence of endochondral bone formation. The animals were decapitated and the muscles were detached from the bones. The occipital bones were detached from the parietal bones at the lambdoidal suture and by two lateral incisions to the foramen magnum. The tibias were removed by cutting through the proximal synovial membranes and joint ligaments without damaging the epiphyseal cartilage. Each tibia was transected at the midshaft, and the proximal half was sectioned longitudinally with a sharp razor blade to obtain a block of tissue approximately 1 mm thick. The blocks of fresh tissues were processed for cytochemical localization of soluble and insoluble calcium phosphate or carbonate salts, and for characterization of the organic matrix associated with the minerals. Staining the f r e s h blocks o fbone with GBHA The fresh blocks of tibia and occipital bone were immersed for three hours in 10% GBHA solution (Kashiwa, '66) to chelate calcium present as soluble and insoluble salts. The 10% GBHA staining solution was prepared by dissolving 0.2 gm GBHA powder to 2 ml of stock solvent containing 3.4% NaOH in 75% ethanol. The stained tissues were immersed in three changes of 100% ethanol to remove the excess GBHA and to dehydrate the tissues. The tissues were then cleared in xylene, and embedded in paraffin. The paraffin blocks were sectioned 7 thick. The sections were floated o...

Direct observation of unstained, 1 mm thick blocks of fresh epiphyseal cartilage from tibia of 15- and 18-day-old chick embryos revealed shrunken chondrocytes on its cut surfaces but unshrunken chondrocytes deep within the tissue blocks. The unshrunken hypertrophied chondrocytes are rimmed with refractile substance identified as chondroitin sulfate removable with hyaluronidase. This substance is stained metachromatically red with toluidine blue, and is stained with ruthenium red and with ruthenium red-OsO4. The latter, observed with the electron microscope, is present as an electron dense rim, specifically about the unshrunken, hypertrophied chondrocytes between the plasma membrane and lacunar wall. By rendering the chondroitin sulfate electron dense with RR-OsO4, electron lucent bodies (ELB) were revealed specifically about the hypertrophied chondrocytes. The ELB contain an electron dense core with radiating fibrils. The content and source of ELB, also found in the intercellular matrix, are not known. The 0.1% toluidine blue solution containing 0.2 M MgC12 or 0.4% NaCl or KCl stained juxtanuclear clusters of granules metachromatically red. The location of intracellular granules was believed to represent a cluster of Golgi-derived vesicles. The pericellular metachromatic, RR-OsO4-positive rim is believed to be an accumulation of externalized juxtanuclear metachromatic granules. The possibility that the ELB may also be externalized content of Golgi vesicles was entertained.