Fig. 2. Specific localization of GFP::Inv in the primary cilia of cultured fibroblasts. The subcellular localization of Inv was examined in primary fibroblasts established from Inv/Inv, Inv::GFP transgenic mice (A-I) or from wild-type (non-transgenic) mice (J-L). (A-C) Nonfixed cells were examined for GFP fluorescence (A) and by differential interference contrast (DIC) microscopy (B); the merged image of A and B is shown at higher magnification in C. GFP fluorescence was associated with rod-like structures (arrowheads) at the periphery of the nucleus (Nu). Scale bar: 5 µm. (D-I) Cells were fixed and subjected to double immunofluorescence staining with antibodies to GFP (red; D,G) and antibodies to acetylated tubulin (green; E,H). (F) Merged image of D,E. (G,H) Higher magnification views of D,E, respectively. (I) DIC image corresponding to G,H. The Inv::GFP fusion protein was specifically localized to primary cilia that were positive for acetylated tubulin (arrowheads). (J-L) Double staining with antibodies to Inv (red, J) and antibodies to acetylated tubulin (green, K). (L) Merged image of J,K. The endogenous Inv protein was also detected in the primary cilia of wild-type fibroblasts (arrowheads).