Intracellular localization of oligonucleotides: influence of fixative protocols

In many studies reporting the use of antisense oligonucleotides (ODN), the intracellular localization was investigated by using fluorescent-labeled oligonucleotides (F-ODN). More often, cells were fixed on uptake of F-ODN before microscopic analysis. We report here the influence of various methods of cell fixation on the intracellular localization of ODN. By confocal microscopy, we show that with unfixed cells, endocytosed peptides, oligonucleotides (Mr around 10,000), and endocytosed proteins were mainly localized in vesicular compartments. On mild fixation with paraformaldehyde, an identical intracellular localization was observed repeatedly after fixation, from immediately up to several days. In contrast, with methods based on the use of strong fixatives, such as methanol or acetone, the small molecules diffuse into the cytosol and in the case of oligonucleotides into the nucleus. These results point out the importance of the fixation protocol in the study of intracellular localization of ODN and their derivatives.