Cells were grown on a coverslip, fixed in 4% paraformaldehyde for 15 min at room temperature, followed by incubation at room temperature for 30 min in 0.5% Triton X-100. The coverslip was washed and incubated with blocking solution for 1 h at room temperature. Primary antibody (1:2,000 dilution, 53BP1, NB100-304, Novus Biologicals) in blocking solution was added, cells were incubated overnight at 4 °C, washed with blocking solution, and then incubated with blocking solution containing DyLight 488 or 549 conjugated secondary antibody for 1 h at room temperature. The coverslip was washed, incubated in 4% paraformaldehyde for 10 min, washed in ethanol series solutions, denatured at 85 °C for 3–5 min, hybridized with PNA probe (Panagene) for 2 h at 37 °C, washed and mounted with DAPI stain and visualized using a Zeiss microscope. Antibodies/probes: rabbit polyclonal anti-53BP1 antibody (Novus), Cy3-labelled CCCTAA, PNA probe (Panagene, Korea).
Pna probe
Manufactured by Panagene
The PNA probe is a synthetic DNA/RNA analogue that can be used to detect and quantify specific nucleic acid sequences. It consists of a peptide backbone with nucleic acid bases attached, which allows it to bind to complementary DNA or RNA strands. The PNA probe can be used in various molecular biology applications, such as gene detection, diagnosis, and research.
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Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Nb100 304, by Novus Biologicals (1 mentions)
Colcemid, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Axiocam, by Zeiss (1 mentions)
Axiovision, by Zeiss (1 mentions)
Ti u microscope, by Nikon (1 mentions)
Pepsin solution, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Texas red, by Panagene (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Cool cube 1 camera, by MetaSystems (1 mentions)
12 protocols using pna probe
1
Immunofluorescence Assay for 53BP1 and Telomeres
2
Quantitative Telomere Length Analysis
3
Fluorescent PNA Probe Synthesis
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The PNA probe was synthesized by Panagene, Inc. (Daejeon, Korea) and the DNA oligomer were purchased from Bioneer (Daejeon, Korea). All PNA probes were 22-mer in length and were labeled with FAM fluorescent dye. The sequences of the DNA oligomer were 5′-AAGACAAAATAACAAAAAGACC-3′ (FAM-PNA-BC1–1 complementary oligomer) and 5′-AACAAGGTAACTGGCACACACA-3′ (FAM-PNA-BC1–3 complementary oligomer).
4
Telomere FISH and MRE11A Binding Analysis
5
Quantifying Telomeric DNA Damage Foci
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After deparaffinization, rehydration, and antigen retrieval, tissue sections were incubated with PNA probe (800 ng/ml, Panagene Inc., Daejeon, Korea) diluted in hybridization buffer (for 5 min at 80°C, then for 2 h at 37°C). After hybridization, sections were washed, incubated with blocking buffer, and finally stained for 53BP1, as described23 (link). To analyze the relationship between DNA damage foci and uncapped telomeres, potential colocalization of 53BP1-foci (IFM) and telomere signal (FISH) was quantified in epidermal cells by visual inspection. For every skin sample, at least 400 basal cells and/or 40 53BP1 + basal cells were analyzed for telomeric 53BP1-foci.
6
Telomere Quantification by Fluorescence In Situ Hybridization
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Telomere length and function (telomere integrity and chromosome stability) was estimated by telomere quantitative FISH. Cells were incubated with 0.5 μg/mL nocodazole for 1.5 h to enrich cells at metaphases. Chromosome spreads were made by a routine method. Metaphase-enriched cells were exposed to hypotonic treatment with 75 mM KCl solution, fixed with methanol: glacial acetic acid (3:1) and spread onto clean slides. Telomere FISH and quantification were performed as described previously [25 (link)], except for FITC-labeled (CCCTAA) peptide nucleic acid (PNA) probe used in this study. Telomeres were denatured at 80 °C for 3 min and hybridized with telomere specific PNA probe (0.5 μg/mL) (Panagene, Daejeon, Korea). Chromosomes were counter-stained with 0.5 μg/mL DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss microscope with FITC/DAPI filters, using AxioCam and AxioVision software 4.6. For quantitative measurement of telomere length, telomere fluorescence intensity was integrated using the TFL-TELO program (gift kindly provided by P. Lansdorp), and calibrated using standard fluorescence beads.
7
Metaphase Fluorescence In Situ Hybridization
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Metaphase fluorescence in situ hybridization was performed using an Alexa488-OO-5′-(CCCTAA)3-3′ (telomeric sequence) and a TMR-OO-5′-CTTCGTTGGAAACGGGA-3′ (centromeric sequence) PNA probe (Panagene). Images were acquired with a Nikon Ti-U microscope using a × 60 objective. All image files were mixed and randomly assigned coded names to allow blinded scoring for chromosome fusions, signal-free ends and fragile telomeres.
8
Telomere Length Determination in Chondrocytes
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Genomic DNA was extracted directly from human chondrocytes using a Mini Genomic DNA Kit (QIAamp DNA Mini Kits) according to the manufacturer’s protocols (Qiagen). Telomere length was determined by using an RT-qPCR method. Telomere FISH was performed by using a PNA probe (Panagene). Briefly, chondrocytes were added to six-well culture plates with glass slides and incubated at 37 °C for 2 h. Adhered cells were swollen in KCl buffer, fixed in methanol/acetic acid (3:1), rehydrated in PBS, fixed in 4% formaldehyde, and then dehydrated in a series of concentrations of ethanol. Slides were incubated with a hybridization mixture (70% formamide, 10 mM NaHPO4, pH 7.4, 10 mM NaCl, 20 mM Tris buffer, pH 7.5), placed on an 80 °C heating block for 5 min to denature chromosomal DNA, and incubated with the PNA probe for 2 h at room temperature. After washing, slides were mounted with Vectashield mounting medium containing DAPI (Vector Labs) and analyzed with a confocal microscope.
9
Telomere FISH Analysis of Adherent Cells
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Telomere FISH was performed by using a PNA probe (Panagene) described as follows65 (link),66 (link). Peritoneal suspensions were collected and were adjusted to 1 × 106 cells ml−1 in DMEM. Then the cells were added to a six-well culture plates with glass slides and incubated at 37 °C for 2 h to allow adherence of macrophages to the glass slides surface. After the incubation, the supernatants containing nonadherent cells (mainly lymphocytes) were removed by gently washing three times with warm PBS. Adhered cells were swollen in KCl buffer (12.3 mM HEPES, 0.53 mM EGTA, 64.4 mM KCl), fixed in methanol/acetic acid (3:1), rehydrated in PBS, fixed in 4% formaldehyde and then dehydrated in a series of concentrations of ethanol. Slides were incubated with hybridization mixture (70% formamide, 10 mM NaHPO4, pH 7.4, 10 mM NaCl, 20 mM Tris buffer, pH 7.5), placed on a 80 ℃ heating block for 5 min to denature chromosomal DNA and incubated with the PNA probe (0.05 mg ml−1) for 2 h at room temperature. After washing, slides were mounted with Vectashield mounting medium containing DAPI (Vector Labs) and analyzed with a confocal microscope (Zeiss LSM 780).
10
Quantifying Telomere Length and Telomerase Activity
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After deparaffinization, tissues were postfixed in 4% formaldehyde. Tissues were incubated at 37 °C for 15 min in pepsin solution (Sigma Aldrich). The tissue sections were mounted on slides and dehydrated in ethanol. After 10 min of air drying, 0.5 mg/ml PNA probe (Panagene) were added to each slide. The slides were incubated for 3 min at 90 °C and for an additional 2 h at room temperature in the dark. Then, the slides were incubated with DAPI (Sigma Aldrich). Confocal images were acquired as stacks using a Leica SP5-MP confocal microscope and maximum projections were done with the LAS-AF software. Telomere signal intensity was quantified using Definiens software. Fifty images per sample were captured. TL values were analyzed using individual telomere spots (300,000 telomere spots per sample). The average fluorescence intensities represent the TL of each sample [15 (link)].
Telomerase activity was quantified with a modified fluorescence telomere repeat amplification assay according to the protocol of the Telomerase Activity Quantification qPCR Assay Kit (KGA1028R, Keygen).
Telomerase activity was quantified with a modified fluorescence telomere repeat amplification assay according to the protocol of the Telomerase Activity Quantification qPCR Assay Kit (KGA1028R, Keygen).
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