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Immunogold Labeling of Amelogenin in Developing Porcine Enamel Revealed by Field Emission Scanning Electron Microscopy

机译：场发射扫描电子显微镜揭示猪牙釉质中釉蛋白的免疫金标记

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摘要

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.
机译：本研究描述了一种使用免疫组织化学标记与高分辨率成像（场发射扫描电子显微镜）相结合的方法来研究发育中的猪瓷釉中牙釉蛋白在磷灰石微晶上的空间定位的方法。用抗重组小鼠牙釉蛋白的抗血清处理来自冻裂猪第三磨牙的发育中釉质组织的横截面，并通过蛋白质印迹分析确认免疫反应性。然后将样品用缀有10 nm金颗粒的山羊抗兔IgG处理。对照样品仅用二抗处理。透镜内二次电子检测器和象限反向散射检测器用于揭示搪瓷结构和金颗粒分布的高分辨率形态。免疫标记显示金颗粒优先沿着带状磷灰石晶体的侧面定位。釉蛋白原在体内在釉质晶体上的优先定位强烈支持其直接作用于控制晶体形态。

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