*2.8. Immunofluorescence Analysis*

CaCo-2 and CoLo-205 cells were seeded into four-well slides chambers at a density of 10 <sup>×</sup> <sup>10</sup><sup>3</sup> cells per well at 37 ◦C and treated with free 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs, respectively, for 24 or 48 h. Untreated cells were used as control. Subsequently, treated and untreated cells were washed three times with PBS, fixed with cold ethanol (96◦ ) for 25 min and permeabilized with an aqueous solution of Triton X-100 (0.5%) in PBS for 15 min. Then cells were blocked with an aqueous solution of normal serum (5%) in PBS for 1 h and incubated at 4 ◦C over-night with the two primary antibodies mix. The protocol applied has been previously described [19], with the following antibodies: mouse monoclonal anti human Vimentin (diluted 1:250 in Blocking) and rabbit polyclonal anti human phospho-Paxillin (Tyr118) (diluted 1:200 in Blocking). Then, cells were washed twice with PBS and incubated with a specific green-fluorescent (goat anti-mouse IgG (H + L) secondary antibody Alexa Fluor 488 conjugate) and red-fluorescent (goat anti-rabbit IgG (H + L) superclonal secondary antibody, Alexa Fluor 555) secondary antibodies for 1 h, at room temperature, in the dark. After washing with PBS, the cells were mounted using prolonged gold antifade reagent containing DAPI (blue). Images were acquired using the Nikon confocal microscope Eclipse Ti2 and the fluorescence intensity was quantified using Image J software (number of pixels/area). Five different areas from the single well for each single independent experiment performed in triplicate were randomly selected [26].
