Fig. 6. Localization of endogenous Frizzled to the actin cytoskeleton and its redistribution in Saos-2 cells expressing Pax3. Confocal images of methanol/acetone-fixed (A-C) and paraformaldehyde-fixed (D-K) Saos-2 cells treated as indicated. In control cells (A,B,D) and cells expressing Pax3^flag (C,E), Frizzled localized to the tips of protrusions, at sites resembling focal adhesions, and in a punctate diffuse manner throughout the cytoplasm. In control cells Frizzled signals overlapped with those of F-actin (detected by phalloidin staining) along stress fibers (arrow in F-G), with prominent Frizzled signals at the ends of stress fibers (H). Cells expressing Pax3^flag have fewer stress fibers (J) relative to control cells (G), and in paraformaldehyde-fixed Pax3-expressing cells, an accumulation of Frizzled signals to cytoplasmic vesicles is apparent (arrowheads in E and I). Cells in F-K were fixed with a paraformaldehyde-cytoskeletal fixation procedure (see Materials and Methods). Bars, 20 µm.