Double immunofluorescence labeling of ZO-1 and Cx45 in HeLa cell cultures. A, Control empty vector-transfected HeLa cells showing labeling of ZO-1 at cell-cell contacts (A1, arrows), and an absence of labeling for Cx45 (A2). B, HeLa cells stably transfected with Cx45. Immunolabeling is seen as linear strands of ZO-1-puncta (B1) and Cx45-puncta (B2) at cell-cell contacts (arrows), and as collections of puncta within cells (arrowheads). ZO-1/Cx45 co-localization is evident at both locations (B3). C, Confocal double immunofluorescence showing co-localization of punctate labeling for ZO-1 and Cx45 at contacts between Cx45-transfected HeLa cells (arrowheads). Scale bars, A,B, 50 μm; C, 10 μm.