Figure 2.

Cellular localization of Cx43–GFP observed by confocal microscopy. (A) A culture of Cx43–GFP-transfected HeLa cells was excited with 450–490 nm light and viewed at 510–520 nm to reveal the location of GFP. Discrete fluorescence plaques, characteristic of gap junctional staining, were observed along the boundary between two cells (arrows). Fluorescence was also detected in the perinuclear region and most likely represents Cx–GFP being processed through the ER. (B) The same area of cells as shown in A stained with Cy3-conjugated antibodies to Cx43 and illuminated at 540–550 nm and viewed at 580–590 nm. A similar staining pattern was observed. (C) The images in A and B were digitally superimposed. The combination of green and red fluorescence results in yellow fluorescence and demonstrates that the membrane plaques and cytoplasmic areas represent sites of colocalization of GFP and Cy3 fluorescence and confirms that GFP fluorescence indicates the location of expressed Cxs. The plaques in C have a green–yellow appearance caused by the larger contribution of green as compared with red. The cells shown were selected for the large number of plaques expressed. For Ca²⁺ experiments, cells with fewer plaques were used. Image width = 60 μm.