Ariotti Nicholas, Hall Thomas E, Parton Robert G
The University of Queensland, Brisbane, QLD, Australia.
Methods Cell Biol. 2017;140:105-121. doi: 10.1016/bs.mcb.2017.03.002. Epub 2017 Apr 25.
The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques.
绿色荧光蛋白（GFP）及相关蛋白的应用彻底改变了光学显微镜技术。在此，我们描述了一种快速简便的方法，通过电子显微镜（EM），采用模块化方法，利用与抗坏血酸过氧化物酶衍生的APEX2标签融合的小GFP结合肽（GBP），在细胞和组织中定位GFP标记的蛋白。我们提供了一种使用模块化APEX-GBP在培养细胞和斑马鱼中通过光学显微镜和电子显微镜观察GFP标记蛋白的方法。此外，我们详细描述了该技术相对于许多现有相关光学和电子显微镜方法的优势，并证明APEX-GBP很容易应用于现代三维技术。
