Preservation of large-scale chromatin structure in FISH experiments.
The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture.
Hepperger
Claudia
C
Department Biologie II, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2, 82152 Planegg-Martinsried, Germany.
Otten
Simone
S
von Hase
Johann
J
Dietzel
Steffen
S
D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000737:chemistry*; D053595:Embryonic Stem Cells; D005557:Formaldehyde; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal; D016707:Tissue Fixation / Q000379:methods*
D002843:Chromatin; D005557:Formaldehyde