Cells on coverslips (70–90% confluency) were processed for antibody detection of NR2F2 or NR2C2 with visualization using fluorescence conjugated secondary antibodies (Anti IgG-Alexa488 ab150113, Abcam, UK). Telomeres were detected by in situ hybridization to Cy3–OO-(CCCTAA)3 PNA probe (PANAGENE) in PNA hybridization solution (70% deionized formamide, 0.25% blocking agent (PerkinElmer), 10 mM Tris–HCl, pH7.2)6 (link). Coverslips were mounted with ProLong Gold containing 5 μg/ml of DAPI. APBs were detected by colocalisation of PML (PML primary antibody, sc-966, Santa Cruz Biotechnology, USA; secondary antibody Alexa488 ab150073, Abcam, UK) with telomeres58 (link).
Cy3 oo ccctaa 3 pna probe
Manufactured by Panagene
Cy3–OO-(CCCTAA)3 PNA probe is a synthetic DNA-like molecule composed of peptide nucleic acid (PNA) with a cyanine 3 (Cy3) fluorescent label. The core function of this probe is to hybridize with complementary DNA or RNA sequences containing the (CCCTAA)3 repeat, which is commonly found in telomeric regions of chromosomes.
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3 protocols using cy3 oo ccctaa 3 pna probe
1
Visualizing Telomeres and APBs in Cells
2
Quantitative Telomere FISH Analysis
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Quantitative fluorescence in situ hybridization (FISH) was carried out as described previously, using Cy3-O-O-(CCCTAA)3 PNA probe (Panagene) (29 (link)). A detailed protocol is provided in Supplementary methods . Images were acquired using Zeiss Axioplan 2. Telomeric signals were quantified with iVision software (Chromaphor) and corrected for the average local background. Statistical analyses were done using the Wilcoxon rank-sum test.
3
Telomere Damage Quantification in Murine Fibroblasts
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Primary murine embryonic fibroblasts (MEFs) were fixed with 2% paraformaldehyde for 15 min followed by permeabilization (0.2% Triton X-100in PBS) for 15 min. Cells were then blocked (2% BSA, 20% goat serum in PBS) for 2 h. Cells were immuno-stained with mouse anti-γH2AX monoclonal antibody (1:500; Upstate, Billerica, MA) overnight and goat anti-mouse 594 secondary antibody (1:1000) for 1 h. Cells were then fixed in 2% paraformaldehyde for 5 min. Samples were dehydrated in 70%, 95%, 100% ethanol (5 min each) and then denatured for 10 min at 80 °C in hybridization solution (70% deionized formamide, 10% NEN blocking reagent [Roche], 0.1 M Tris-HCl [pH 7.4], MgCl2 buffer [82 mM NaH2PO4, 9 mM citric acid, 20 mM MgCl2], and 0.5 µg/mL Cy3-OO-(CCCTAA)3 PNA probe (Panagene, South Korea). After 2 h hybridization at room temperature, the samples were washed twice with 70% deionized formamide in 10 mM Tris-HCl, pH 7.2. Samples were counterstained with DAPI, mounted onto slides with Gelvatol and images were acquired with a Nikon A1 confocal microscope (Nikon Instruments, Inc.).
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