Fig. 2. Caveolin-1-GFP is located in caveolae. (A) A mixed culture of parental CHO cells and CHO cells stably expressing the caveolin-1-GFP were plated on coverslips and grown for 2 days before they were fixed in methanol and labeled with anti-caveolin-1 followed by an Alexa-568-labeled goat anti-rabbit IgG. The green channel (GFP) shows only those cells that express caveolin-1-GFP, while the red channel shows anti-caveolin-1 staining. The anti-caveolin-1 antibody detects both endogenous caveolin-1 and caveolin-1-GFP. The merged image shows nearly perfect overlap indicating that all of the GFP signal is from caveolin-1-GFP and not from GFP that has been cleaved from the caveolin. The addition of a GFP moiety has not affected the distribution of caveolin (compare the caveolin distribution in the non-expressing cell at the top with one that is expressing the GFP construct below it). (B) Cells were grown for 2 days in 6-well tissue culture dishes and incubated for 1.75 hours in the presence of 10 µM nocodazole, which was used to enhance our ability to recognize caveolae at the cell surface of these cells. The samples were then fixed and processed for immunogold localization of GFP. Bars, 20 µm (A); 0.25 µm (B).

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