The distribution of calcium in undecalcified bone as revealed by an improved pyro-antimonate method

The localization of pyro-antimonate-precipitable Ca2+ in the undecalcified femur and calvaria of neonatal rats was examined. The fixation of bones with pyro-antimonate-glutaraldehyde followed by pyro-antimonate-osmium (two-step method) resulted in better preservation of tissue and more precise localization of precipitates than did the direct immersion of specimens in pyro-antimonate-osmium solution (one-step method). The precipitate was frequently observed within the endoplasmic reticulum of obsteoblasts. Most vacuoles in osteoclasts contained precipitate. By contrast, the mitochondria in these cells were associated with small amounts of precipitate. There was no evidence of precipitate in the Golgi apparatus. The presence of calcium in the precipitate was verified by EGTA treatment and X-ray microanalysis. This study demonstrated that (1) the two-step pyro-antimonate method is a useful and reliable procedure for visualizing Ca2+, and (2) cellular Ca2- can be successfully localized in undecalcified bone by this method.