Figure 2.

Immunolocalization of Cx43 GJs and HCs in cultured human gingival fibroblasts. (A–C) Representative images from GFBL-DC cultures fixed and permeabilized with 0.5% Triton X-100 treatment before double immunostaining with antibodies against all forms of Cx43 (red) and ZO-1 (green), indicator of cell-cell contacts. Fibroblasts displayed numerous Cx43-positive plaque-like structures throughout the cell body. Some of these plaques colocalized with ZO-1 staining at apparent cell-cell contacts and over the cell body (arrowheads in A), likely representing GJ plaques. However, Cx43-positive structures that did not colocalize with ZO-1 (arrows) were also present. (D–F) Representative images of confluent GFBL-DC cultures fixed and permeabilized with 0.5% Triton X-100 treatment before double immunostaining with Cx43(E2) antibody specific for Cx43 HCs (red) and ZO-1 (green). Cx43 HCs were also organized in plaque-like structures present along the cell body, but they were not colocalized with ZO-1 present in the cell-cell contact areas (arrowheads in D). Some cells showed localization of ZO-1 in the nucleus consistent with its function also as a transcription factor. (G–I) Representative images of a confluent gingival fibroblast (GFBL-DC) culture fixed and then double immunostained with the Cx43(E2) (red) and ZO-1 (green) antibodies without permeabilization with 0.5% Triton X-100 to detect only cell surface-associated Cx43 HCs. In these cells, the Cx43(E2)-positive HC-plaques localized along the cell body, but their number was reduced compared to the permeabilized cells (D and E). No immunoreactivity for the intracellular ligand ZO-1 was detected as expected (I). Nuclear staining (blue) was performed using DAPI. Magnification bar = 10 μm.