Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization.
We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci.
Hendzel
M J
MJ
Department of Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta, Canada T2N 4N1.
Bazett-Jones
D P
DP
D000107:Acetylation; D002843:Chromatin / Q000737:chemistry / Q000648:ultrastructure*; D004247:DNA / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D000432:Methanol / Q000494:pharmacology; D009997:Osmotic Pressure; D011108:Polymers / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification; D016707:Tissue Fixation / Q000379:methods*
D002843:Chromatin; D005404:Fixatives; D006657:Histones; D011108:Polymers; D005557:Formaldehyde; D012313:RNA; D004247:DNA; C003043:paraform; D000432:Methanol