Calcium in Cells of Fresh Bone Stained with Glyoxal Bis(2-Hydroxyanil)

Abstract

Fresh ileum of adult rats and vertebrae and calvariae of newborn rats were immersed in a staining solution containing 0.1 gm of glyoxal bis(2-hydroxyanil) (GBHA) per 2 ml of 3.4% NaOH in 75% ethanol, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Paraffin sections of stained material, 7 μ thick, were affixed to albumenized slides, immersed in 90% ethanol saturated with Na₂CO₄ and KCN to ensure specificity for calcium, rinsed in 95% ethanol, counterstained in 50% ethanol containing 0.1% methylene blue, dehydrated in absolute ethanol, deparaffinized and cleared in xylene, and mounted in neutral synthetic resin. By this procedure, red Ca-GBHA granules were deposited in goblet and Paneth cells, and in the cytoplasm of osteoblasts, osteocytes, chondrocytes, and periosteal cells of developing bones. Calcium in apatite did not stain. In osseous tissues sectioned in a cryostat or processed by the freeze-dry or freeze-substitution method, epiphyseal chondrocyte calcium was removed, and apatite stained so intensely red that it obscured calcium in the bone cells. Failure of control osseous tissues to stain after immersion in a 1% solution of disodium salt of ethylenediaminetetraacetic acid or in the alcoholic, alkaline solvent of the GBHA solution, indicated that the red granules in the cells of developing bone were due to calcium present in the cells in vivo and not due to absorption of GBHA by tissue components other than calcium, or to diffusion of Ca⁺⁺ during staining. Calcium localized in the cytoplasm and processes of the osteogenic cells suggests the need to re-evaluate the role of osteoblasts as depositors of calcium during osteogenesis.