Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.
Methods Mol Biol. 2021;2346:191-206. doi: 10.1007/7651_2020_330.
Gap junction (GJ) research has entered a new stage focusing the concerted dynamic behavior of multiple isoforms of connexin (Cx) in the cell membrane, cytosolic vesicles, and space between them. To proceed with this research, imaging technologies are important. Here we describe two novel protocols for this purpose. At first, the adoption of a small motif of Cys-Cys-X-X-Cys-Cys as a visualization tag is described. An As complex, FlAsH, can bind to this tetra-Cys (TC) tag to form a fluorescent conjugate. Its introduction into the C-terminal of Cx43 is demonstrated. Next, a novel triangle chip for the accurate x-y registration is described. Target single cells of HeLa marked with a fluorescent dye can be easily recognized by electron microscopy based on this chip.
缝隙连接（GJ）研究已经进入了一个新阶段，重点研究细胞膜、胞质小泡及其间隙中多种连接蛋白（Cx）的协同动态行为。为了进行这项研究，成像技术非常重要。在这里，我们为此目的描述了两种新的方案。首先，描述了采用 Cys-Cys-X-X-Cys-Cys 小基序作为可视化标签的方法。一种 As 配合物，FlAsH，可以与这个四半胱氨酸（TC）标签结合形成荧光缀合物。将其引入 Cx43 的 C 端进行了展示。接下来，描述了一种新颖的三角形芯片，用于精确的 x-y 配准。使用这种芯片，通过电子显微镜可以很容易地识别用荧光染料标记的 HeLa 靶标单细胞。
