Strand-specific chromatid orientation-fluorescence in situ hybridization (CO-FISH) was performed according to Bailey et al. (2004) (link), with minor modification. Subconfluent cells were incubated with BrdU. Nocodazole was added for 1.5 hr prior to cell harvest, and metaphase spreads were prepared by standard cytogenetic method as described earlier. Chromosome preparations were stained with Hoechst 33258 (0.5 μg/ml), incubated in 2× SSC (Invitrogen) for 20 min and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 40 min. The BrdU-substituted DNA was digested with Exonuclease III (Promega). The slides were then dehydrated through cold ethanol series and air dried. PNA-FISH was performed with Fluorescein-OO-(CCCTAA)3 (Bio-Synthesis). Slides were hybridized, washed, dehydrated, mounted, and counterstained with VectaShield antifade medium (Vector), containing 0.1 μg/ml DAPI. Digital images were captured using a CCD camera on a Zeiss Axio-Imager Z1 microscope equipped with Metasystems Isis software.
Fluorescein oo ccctaa 3
Manufactured by Bio-Synthesis
Sourced in United States
Fluorescein-OO-(CCCTAA)3 is a chemical compound used in various laboratory applications. It consists of a fluorescein molecule linked to a specific DNA sequence, (CCCTAA)3. This product can be utilized for various research purposes, but a detailed description of its intended use would require further information.
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2 protocols using fluorescein oo ccctaa 3
1
Strand-specific chromatid orientation by CO-FISH
2
Telomere Length Measurement by CO-FISH
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CO-FISH assay was performed according to our previous work [24 (link)]. Briefly, subconfluent cells were incubated with 10 μM of 5′-bromo-2′-deoxyuridine (BrdU) for 12 h to allow BrdU incorporation for one cell cycle. Nocodazole was added for 2 h prior to cell harvest, and metaphase spreads were prepared by standard cytogenetic method as described above for Q-FISH. Chromosome slides were treated with RNaseA, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/mL) for 15 min and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 40 min. The BrdU-substituted DNA was digested with Exonuclease III (Promega, Madison, WI, USA). The slides were then dehydrated through cold ethanol series and air dried. PNA-FISH was performed with Fluorescein-OO-(CCCTAA)3 (Bio-Synthesis, Lewisville, TX, USA). Slides were hybridized, washed, dehydrated, mounted, and counterstained with VectaShield antifade medium (Vector, Peterborough, UK), containing 0.1 mg/mlDAPI. Digital images were captured using a CCD camera on a Zeiss Axio-Imager Z1 microscope equipped with Metasystems Isis software.
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