PDF(Pubmed)
Abstract：
The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.
摘要：
初级纤毛上蛋白质定位的明确证明一直是纤毛生物学家的挑战。初级纤毛是孤立的线状突起，具有特殊的蛋白质组成，但是纤毛结构覆盖细胞膜和其他细胞部分，纤毛蛋白的身份很难通过常规成像方法如免疫荧光显微镜来确定。表面扫描电子显微镜与免疫标记（免疫SEM）相结合，通过在纤毛的三维超微结构的背景下明确显示蛋白质表达，绕过了其中一些不确定性。这里,我们应用免疫SEM特异性鉴定小鼠和人胰岛初级纤毛上的蛋白质，包括翻译后修饰的微管蛋白，步内运输（IFT）88，小GTP酶Arl13b，以及轴突动力蛋白的亚基。样品制备中的关键参数，讨论了免疫标记和成像采集，以促进纤毛研究界其他人的类似研究。
