Ultrastructural immunolocalization of a major phosphoprotein in embryonic chick bone

Immunocytochemistry utilizing the protein A-gold technique was used to examine the ultrastructural cellular and extracellular distribution of a major phosphoprotein in chick bone. HCl-extracts of embryonic and neo-natal chick bones contain a major 66kD phosphoprotein (BPP) which was purified and used to raise polyclonal antibodies in rabbits. The mid-diaphyseal regions of 8-, 12- and 18-day embryonic chick tibiae were fixed with 1% glutaraldehyde and embedded in Epon or Lowicryl. Electron microscopy following incubation of tissue sections with the antibody and the protein A-gold complex revealed specific immunolabeling over the rER and Golgi apparatus of osteoblasts and over those areas of bone matrix containing Ca and P as determined by electron probe x-ray microanalysis. These included extracellular areas in the matrix undergoing early mineralization and electron dense patches occurring at the mineralization front and extending throughout the more mature bone regions. Biochemical analyses of bone tissue processed similarly to that used for immunocytochemistry confirmed the retention of phosphoprotein in the tissue. The spatial correlation of phosphoprotein in the extracellular matrix with Ca-P mineral deposits confirms an earlier report using 33Pi and radioautography and may indicate a role for phosphoproteins in calcification.