Influence of cell fixation on chromatin topography

Using in situ hybridization techniques, a fixation step must precede denatu ration to prevent disintegration of the chromosomes. The analysis of nuclei fixed by paraformaldehyde, preserving the native structure (three-dimensio nal or 3D fixation and analysis) has become possible with the development o f confocal microscopy; however, the analysis of those fixed by methanol and acetic acid, dehydrating the nuclei (two-dimensional or 2D fixation and an alysis), remains a very valuable tool for practical use in diagnostics and also in many cases for research. We compared both types of fixation and ana lyses using different cell lines and different DNA probes. Fixation of cell s by methanol and acetic acid leads to the enlargement of contact of nuclei with the slide surface, resulting in a substantial increase of nuclear dia meter, flattening of the nucleus, and consequently to a distortion of gene topology. Our results indicate that chromatin structures located in the out er parts of the nuclear volume (e.g., heterochromatin of some centromeres) are relatively shifted to the membrane of these nuclei, keeping the absolut e centromere-membrane distance constant. On the other hand, euchromatin loc ated in the inner parts of the nuclear volume is not shifted outside propor tionally to the increase of molecular dimensions; consequently, the relativ e distances for the center of nucleus to gene are smaller after methanol-ac etic acid fixation. The limitations of the analysis of dehydrated preparati ons for practical use and in research are discussed. (C) 2000 Academic Pres s.