Cells were incubated with 0.5 μg/ml nocodazole (sigma) for 6 h to enrich cells at metaphases. Metaphase-enriched cells were hypotonic treated with 75 mM KCl solution, fixed with methanol∶glacial acetic acid (3:1), and spread onto clean slides. Telomeres were denatured at 85°C for 4 min and hybridized with Cy3-labeled (CCCTAA)3 PNA probe (Panagene). Chromosomes were stained with DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss Axion Imager Z1 microscope. For quantitative measurement of telomere length, telomere fluorescence intensity was integrated using the TFL-TELO program.
Axion imager z1 microscope
Manufactured by Zeiss
The Axion Imager Z1 is a high-performance microscope designed for advanced imaging applications. It features a modular design, allowing for the integration of various imaging techniques and accessories. The microscope provides precise control and versatile imaging capabilities to support research and analysis in a wide range of scientific fields.
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4 protocols using axion imager z1 microscope
1
Measuring Telomere Length by PNA FISH
2
Quantitative Telomere Length Determination
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Cells were treated with 0.5 μg/ml nocodazole (sigma) for 6 h to enrich cells at metaphase. Metaphase-enriched cells were hypotonic with 75 mM KCl solution, fixed with methanol/glacial acetic acid (3:1), and spread onto clean ice-cold slides. Telomeres were denatured at 85°C for 5 min and hybridized with Cy3-labeled (CCCTAA)3 PNA probe (Panagene). Chromosomes were stained with DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss Axion Imager Z1 microscope. For quantitative measurement of telomere length, telomere fluorescence intensity was integrated using the TFL-TELO program.
3
EdU Incorporation Visualization in Cells
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MRC5 and BJ cells on the coverslip were cultured with medium supplemented with 10 μM EdU for indicated times (MRC5 cells for 2 h and BJ cells for 4 h, respectively). Then, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Following incubation, the fixed cells were permeabilized with 0.3% Triton X-100 (in 1× PBS) for 15 min at room temperature. Cells were stained at room temperature for 30 min with staining buffer (10 μM FAM-azide, 1 mM CuSO4 and 10 mM sodium ascorbate in PBS), then washed and mounted with DAPI. Fluorescence was detected and imaged using Zeiss Axion Imager Z1 microscope.
4
Telomere Structure Visualization via Immunostaining
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Briefly, cells on the coverslip were fixed with 4% paraformaldehyde, then permeabilized with 0.5% Triton X-100 (in 1 × PBS). Cells were incubated overnight at 4°C with primary antibodies against γH2AX (1:200 dilution, 2577, Cell Signaling Technology), PCNA (1:200 dilution, GTX100539, Genetex), RPA1 (1:200 dilution, sc-28304, Santa Cruz), TRF2 (1:200 dilution, 05–513, Merck), or TRF1 (1:50 dilution, GTX77605, Genetex), washed three times with 1 × PBST, and incubated with secondary antibodies (1:2000 dilution, DyLight488-conjugated anti-mouse or anti-rabbit, KPL). Cells were washed three times with 1 × PBST, re-fixed with 4% paraformaldehyde for 30 minutes, dehydrated by ethanol series solution, denatured at 85°C for 5 minutes, and then hybridized with Cy3-labeled (CCCTAA)3 PNA probe (Panagene) at 37°C for 4 hours. The cells were washed and mounted with DAPI. Fluorescence was detected and imaged using Zeiss Axion Imager Z1 microscope.
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