Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones

Polyclonal antibodies against a 44-KD phosphoprotein (44K BPP) from rat bone were raised in rabbits, affinity-purified, and used as probes to study the protein's distribution in various types of developing bones from newborn rats. Three immunostaining procedures were applied utilizing indirect immunofluorescence, avidin-biotin-peroxidase complex, and avidin-gold complex with silver enhancement. All methods gave essentially identical and/or complementary results. Antigenicity for anti-44K BPP was detected in endochondral and membranous bone. In the latter, it was also demonstrated in the osteoid. In the woven bone of lower jaw, immunoreactivity for anti-44K BPP antibodies was found in fibroblast-shaped cells (pre-osteoblasts) that were between the bone trabeculae but not in direct contact with bony extracellular material. In addition to these presumed osteoprogenitor cells, osteoblasts as well as osteocytes were strongly stained; the cytoplasmic staining was associated with the Golgi apparatus. Occasionally immunoreactivity was detected in osteoclasts, but in these cells immunostaining was either diffusely spread in the cytoplasm or present only at sites of bone erosion. These findings support the hypothesis that the 44K BPP is a protein made by osteoblasts and is localized predominantly in bone. Furthermore, the protein appears to be expressed early in histogenesis of the bone-forming cells.