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Fig. 7. Immunolocalization of ZO-1 and Src in annular gap junctions and Amira volume-reconstructions. (A) Upper panels: localization of ZO-1 (red) in serial sections (0, 1, 2, 3 µm) of a Cx43-GFP annular gap junction (AGJ) characterized by the presence of green fluorescence. Notice that colocalization (shown in yellow) occurs inside the annular gap junction. Lower panels: localization of Src (red) in serial sections of a Cx43-GFP-containing AGJ (green). Notice that colocalization (shown in yellow) occurs outside the AGJ. (B,C) Amira volume-reconstructions of the interactions between ZO-1 and Cx43, and Src and Cx43 in AGJ. (B) Tilting angles (–45°, 0°, +45°) of an entire AGJ reconstructed with Amira software from Cx43-GFP (green) and ZO-1 (red) experiments demonstrate the absence of ZO-1 outside of the structure (upper panels). When `cutting open' part of the AGJ, ZO-1 clearly appeared accumulated inside (lower panels). Semi-quantitative analysis confirmed the internal localization of ZO-1 (right panel). (C) Amira volume-reconstructions of interaction between Src and Cx43 in annular gap junctions. Tilting angles (–45°, 0°, +45°) of a complete AGJ reconstruct with Amira software from Cx43-GFP (green) and Src (red) experiments demonstrating strict outside localization of the kinase on the AGJ (arrows and insets). Even if some Src signals are present just near the AGJ, i.e. without making contact, others are tightly associated with the AGJ (arrows). Cross sections of rotated open AGJ show no Src signal inside the AGJ. Semi-quantitative analysis confirmed Src labeling at the outside (right panel) versus ZO-1 labeling at the inside. Values are the means ± s.e.m. **P<0.01, n=100.
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