Ultrastructural localization of alkaline phosphatase in initial intramembranous osteogenesis

Developing fetal calvariae in which calcification of woven bone has been initiated, have been decalcified with formic acid, buffered with citrate at pH5 to remove all traces of calcium and phosphate from the tissue. The decalcified bone was then incubated in medium containing sodium-beta-glycerophosphate, an artificial substrate for alkaline phosphatase. The Hugon and Borgers-modified Gomori reaction revealed the sites of A-Pase activity as electron dense deposits of lead phosphate. 23,24 One site of A-pase localization was in Golgi vesicles. Another site, at the cell membrane, indicated that A-Pase was apparently secreted from the osteoblasts into the calcifying matrix. A strong reaction in the matrix vesicles and at the periphery of bone nodules (vesicles) indicated that A-Pase was localized ultrastructurally in the precise zones of calcification during initial intramembraneous ossification.