Optimization of nuclear transcript detection by FISH and combination with fluorescence immunocytochemical detection of transcription factors.
Detection of specific nuclear transcripts by fluorescence in situ hybridization (FISH) has constituted a major breakthrough in the study of the organization of transcription in the cell nucleus. Using the model of heat shock genes, we present an optimized procedure for nuclear transcripts that provides high efficiency for RNA detection and good preservation of cell morphology and nuclear texture. Using this procedure, we designed an original high-efficiency methodology combining FISH and fluorescence immunocytochemistry (FICC), which is used here for the simultaneous detection of heat-shock protein (hsp) nuclear transcripts and the specific heat-shock transcription factor 1 (HSF1). We show that the nuclear accumulation sites of HSF1 in heat-shocked cells do not correspond to the sites of transcription of the hsp70 gene.
Jolly
C
C
DyOGen, INSERM U309, Institut Albert Bonniot, La Tronche, France.
Mongelard
F
F
Robert-Nicoud
M
M
Vourc'h
C
C
D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics; D000076249:Heat Shock Transcription Factors; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012313:RNA / Q000032:analysis; D014157:Transcription Factors / Q000032:analysis*
D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D018840:HSP70 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D014157:Transcription Factors; D012313:RNA; D004247:DNA