*2.6. In vitro Investigation by Field Emission Scanning Electron Microscopy*

CaCo-2 and CoLo-205 cells were seeded on silicon wafers (Ted Pella Inc., Redding, CA, USA) at a density of 2 <sup>×</sup> <sup>10</sup><sup>3</sup> /well in 24-wells plates and at a subconfluent density, exposed to 5-FU/LPs and anti-FZD10/5-FU/LPs at a drug concentration of 2 µM for 6, 24 and 96 h. After cell washing with PBS, all cell lines were fixed with 3% glutaraldehyde in PBS for 1 h at 4 ◦C and incubated in 1% OsO<sup>4</sup> for 1 h. The samples were washed five times with aqueous solution of sodium cacodylate (0.005 M, pH 7.2). Then, sequential cell treatment steps with water/acetone mixtures, gradually increasing the acetone volume from 20% to 100%, were carried out to accomplish the complete dehydration process of the cells. The same fixation procedure was carried out after deposition on silicon chips also for the untreated cell lines. The fixed and dried cells were coated with a uniform Au metal film, a few nanometers thick, that was deposited on the samples placed on silicon chips using a turbomolecular pump SC7620 Mini Sputter/Glow Discharge System, Quorum Technologies, and imaged using a Zeiss Sigma FE-SEM. A constant Extra-High Tension (EHT) value of 3.00 kV and a working distance (WD) ranging from 1.8 to 3 mm were set for the FE-SEM observation. For each experiment and sample, a representative FE-SEM micrograph was selected after the collection of a set of images resulting from three replications of the same experiment [20].
