Influence of different fixation reagents on NIH 3T3 fibroblasts morphology: An AFM study

The Atomic force microscope (AFM) has recently become an attractive tool for biological analysis, especially for the characterization of both morphological and mechanical properties. In this study, we investigated the influence of different fixation reagents on NIH 3T3 fibroblast cells morphology. Three protocols were selected: paraformaldehyde, methanol and acetone. Height, perimeter, area, volume and roughness of cells were measured using an AFM. As expected, different fixation protocols produce different morphological changes in cells. However, to preserve fibroblastsí morphology, the methods applying aldehyde fixations performed much better than those using either methanol or acetone. Based on quantitative assessments, fibroblasts fixed with paraformaldehyde showed the closest volume, area and perimeter values to living cells. Roughness data and a Western blot test confirmed that methanol fixation produces larger cell membrane damage, when compared to the other two fixatives.