Alphaphot 2 ys2

Light microscopy preparations were obtained from 3 individuals of each experimental group by cutting 1e2 mm thick slices of the digestive gland with a razor blade from the gland's surface, close to the kidney's boundary (Koch et al., 2006) . The samples were fixed in 4% paraformaldehyde for 24 h. Then, they were kept in 70% ethanol, subsequently dehydrated in increasing concentrations of alcohol, embedded in Histoplast ® and sectioned (7 mm). Ten separate sections per animal were studied in snails under control, exposure and depuration conditions. Digital micrographs at 100Â magnification were obtained with a color video camera on a microscope Nikon ALPHAPHOT-2 YS2. Both intracellular corpuscular types, C and K, were identified by its natural pigmentation (Castro-Vazquez et al., 2002; Koch et al., 2006) and then were contoured using Image Pro-Plus 6.0 (Media Cybernetics, Silver Spring, MA, USA). Glandular symbiotic occupancy was calculated dividing the sum of the total area occupied by each type of symbiotic corpuscle by the acinar area and then multiplying by 100 (Table S1).

Cell uptake of the FUDL-5FU was followed upon incubation on HaCaT and SK-Mel-28 cells by fluorescence microscopy.
Liposomal cell uptake was determined on both cell types grown 24 h on coverslips in 6-well plates. After 3 h of incubation in the dark at 37 °C (optimum temperature of growth) and at 4 °C (temperature at which internalization by endocytic uptake is absent due to reduced metabolism of cells [12] (link)), liposomal suspensions were removed, cells were washed, and coverslips were mounted on a fluorescence microscope (Nikon Alphaphot-2 YS2, Japan). RAW 264.7 cell line was used as a positive control of phagocytosis. The image merge was carried out using ImageJ software (US NIH, Bethesda, Maryland, USA).