Application of reflected light microscopy to identify and quantitate resorption by isolated osteoclasts

A simple technique has been developed to identify the resorption lacunae excavated by avian osteoclasts in vitro. Briefly, devitalized bovine bone wafers, with cells in situ, are fixed, stained with toluidine blue, and then examined by reflected light microscopy. Resorption pits are clearly visible using a x 10 objective, even when the resorbed areas are covered by a confluent layer of cells. The technique can be used to quantify the plan area, depth, and volume of the excavations. Depth and volume are assessed using the x 50 objective lens. This technique has been used to investigate the effects of retinyl acetate and EHDP on bone resorption.