*5.7. Cell Morphology Analysis*

HGEC cells were grown on gelatinized glass coverslips (12 mm) and treated as it was described above. For cell morphology analysis, FabF8:Stx2 were used at 1 μg/mL. Following, cells were fixed with 96% v/v alcohol for 2 h at room temperature and stained with hematoxylin/eosin (H&E). Subsequently, HGEC were analyzed by light microscopy (×200 and ×400, Zeiss Axiophot, Zeiss, Heidelberg, Germany). The percentage of cells/field was obtained from photographs of 10 randomly selected fields. Cells were then counted and averaged, and the percentage of cells per field was estimated by considering the average number of controls as 100% (percentage of cells/field = (number of treated cells × 100)/number of control cells). Furthermore, the percentage of cell area was calculated from the same photographs. For that, the cell area was analyzed in each cell by using the Image J software (NIH) according to the manual instructions. The cell area average was calculated for each condition and the cell area of controls was considered as 100% (percentage of cell area/field = (cell area of treated cells × 100)/cell area of control cells [28]. Results were expressed as means ± standard deviation of the mean (SD). The percentages of protection from cell detachment and intracellular edema were calculated considering 100% prevention when these alterations were totally reversed.
