Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate Registration of Target Single Cells Based on a Triangle Standard Chip for the Image Analysis of Gap Junctional Communication.

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.