THE ANATOMY OF BONE SIALOPROTEIN IMMUNOREACTIVE SITES IN BONE AS REVEALED BY COMBINED ULTRASTRUCTURAL HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY

Bone sialoprotein was immunolocalized at the EM level in thin Lowicryl K4M sections of rat bone. Because of the unconventional EM morphology of the bone matrix seen in thin demineralized acrylate sections, the pattern of immunolabeling was compared with detailed structural images of demineralized bone obtained using an en bloc treatment of tissue s amples with the cationic electron 'dye,' Malachite Green (MG), which p rovides stabilization and retention of anionic material throughout spe cimen processing. A system of structures corresponding to the sites of bone sialoprotein (BSP) immunoreactivity, as seen in Lowicryl K4M thi n sections, could be readily identified in the MG-treated, epoxy thin sections. This system includes the cement lines, and aggregates of sim ilar material within mineralized bone and mineralizing osteoid. The vi rtual identity of BSP distribution with the arrangement of the MG-visu alized material indicates that a BSP-enriched, noncollagenous phase ca n be demonstrated using different, unrelated tissue preparation and im aging protocols for EM. Besides improving our understanding of the dis tribution of bone sialoprotein in bone, these data assign a previously unrecognized structural dimension to noncollagenous material in the b one matrix.