Immunogold labeling of amelogenin in developing porcine enamel revealed by field emission scanning electron microscopy.
The present study describes a method using immunohistochemical labeling in combination with high-resolution imaging (field emission scanning electron microscopy) to investigate the spatial localization of amelogenins on apatite crystallites in developing porcine enamel. Cross-sections of developing enamel tissue from freeze-fractured pig third molar were treated with antiserum against recombinant mouse amelogenin and immunoreactivity confirmed by Western blot analysis. The samples were then treated with the goat anti-rabbit IgG conjugated with 10-nm gold particles. The control samples were treated with the secondary antibody only. The in-lens secondary electrons detector and quadrant back-scattering detector were employed to reveal the high-resolution morphology of enamel structures and gold particle distribution. The immunolabeling showed a preference of the gold particle localization along the side faces of the ribbon-like apatite crystals. The preferential localization of amelogenin in vivo on enamel crystals strongly supports its direct function in controlling crystal morphology.
Du
Chang
C
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, Calif. 90033, USA.
Fan
Daming
D
Sun
Zhi
Z
Fan
Yuwei
Y
Lakshminarayanan
Rajamani
R
Moradian-Oldak
Janet
J
D053523:Amelogenin / Q000378:metabolism*; D000818:Animals; D003743:Dental Enamel / Q000254:growth & development* / Q000648:ultrastructure*; D005614:Freeze Fracturing; D007150:Immunohistochemistry; D008855:Microscopy, Electron, Scanning; D034421:Sus scrofa / Q000254:growth & development*; D014070:Tooth / Q000166:cytology
D053523:Amelogenin