Open AccessQuantitative assessment of the alteration of chromatin during the course of FISH procedures
Author(s) -
Mongelard Fabien,
Vourc'h Claire,
RobertNicoud Michel,
Usson Yves
Publication year - 1999
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19990601)36:2<96::aid-cyto2>3.0.co;2-x
Subject(s) - dapi , chromatin , staining , biology , in situ , fixation (population genetics) , fish <actinopterygii> , denaturation (fissile materials) , dna , in situ hybridization , fluorescence in situ hybridization , confocal , confocal microscopy , microbiology and biotechnology , biophysics , chemistry , biochemistry , genetics , chromosome , optics , gene expression , physics , organic chemistry , fishery , nuclear chemistry , gene
Background: DNA denaturation, required for fluorescent in situ hybridization (FISH) experiments, is likely to induce chromatin alterations. Only few attempts have been made to quantify the extent of these perturbations. We propose a quality‐control approach based on image analysis to monitor the effect of a procedure commonly used in FISH experiments. Methods: Using DAPI as a probe, the same nuclei were successively imaged with a CCD camera after fixation, after permeabilization, and after thermal denaturation and hybridization with a centromeric probe. The modifications of the staining pattern were analyzed. Volumes of the FISH signals were measured using confocal imaging. Results: DAPI staining combined with image analysis proved to be a sensitive tool to visualize the effects of different treatments used in FISH experiments. Permeabilization of nuclei after fixation has only limited impact on the chromatin. On the contrary, the denaturation procedure modifies the staining of DNA by DAPI, as well as the underlying chromatin structure as assessed by the increase of FISH signal volume with denaturation time. The protocol that involves a pre‐fixation permeabilization step results in a more severe loss of chromatin structure. Conclusions: Our results clearly show that analysis of alterations of DAPI staining patterns is a useful monitoring tool to control and standardize hybridization procedures. Cytometry 36:96–101, 1999. © 1999 Wiley‐Liss, Inc.