Quantitative assessment of the alteration of chromatin during the course of FISH procedures

Background: DNA denaturation, required for fluorescent in situ hybridizatio n (FISH) experiments, is Likely to induce chromatin alterations. Only few a ttempts have been made to quantify the extent of these perturbations. We pr opose a quality-control approach based on image analysis to monitor the eff ect of a procedure commonly used in FISH experiments. Methods: Using DAPI as a probe, the same nuclei were successively imaged wi th a CCD camera after fixation, after permeabilization, and after thermal d enaturation and hybridization with a centromeric probe. The modifications o f the staining pattern were analyzed. Volumes of the FISH signals were meas ured using confocal imaging. Results: DAPI staining combined with image analysis proved to be a sensitiv e tool to visualize the effects of different treatments used in FISH experi ments. Permeabilization of nuclei after fixation has only limited impact on the chromatin. On the contrary, the denaturation procedure modifies the st aining of DNA by DAPI, as well as the underling chromatin structure as asse ssed by the increase of FlSH signal volume with denaturation time. The prot ocol that involves a pre-fixation permeabilization step results in a more s evere loss of chromatin structure. Conclusions: Our results dearly show that analysis of alterations of DAPI s taining patterns is a useful monitoring tool to control and standardize hyb ridization procedures. Cytometry 36:96-101, 1999. (C) 1999 Wiley-Liss Inc.