Immunogold labeling of amelogenin in developing porcine enamel revealed by field emission scanning electron microscopy

Abstract

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.

Fig. 1.

a Photograph of a freeze-fractured tooth cusp from a third molar crown of a 6-month-old pig. For biochemical analysis, the enamel tissue was roughly divided into 3 zones (approx. 2 mm in length each) in the direction cervical margin to cuspal edge. b SDS-PAGE pattern (left) and Western blots (right) of acetic acid extract of enamel tissue scrapings from the 3 zones. Lane 1: Bio-Rad Precision Plus standard proteins. Lane 2 and 2′: zone 1, cervical portion. Lane 3 and 3′: zone 2, middle portion. Lane 4 and 4′: zone 3, cuspal portion. Lane 5: Invitrogen SeeBlue Plus2 standard proteins. a–e = Various porcine amelogenin components, presumably 25, 23, 20, 13 kDa and tyrosine-rich amelogenin peptide (TRAP), respectively.

Fig. 2.

FESEM micrographs of the enamel tissue after immunogold labeling. a SE image. b BSE image of the enlargement of the square area in a. c Image recorded by mixing both SE and BSE signals. d SE image of a control sample without the anti-rM179 serum incubation. R = Rod; IR = interrod. Arrows highlight the localization of gold nanoparticles close to the edge or side face of the mineral ribbons.