Immunofluorescence, telomere-fluorescence in situ hybridization (T-FISH) and TERRA RNA-FISH

Cells were pre-extracted with ice-cold CSK buffer [10 mM PIPES pH 8.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 0.5% (v/v) Triton X-100], then fixed with 4% paraformaldehyde for 10 min. The coverslips were blocked with goat serum, then treated with the indicated primary antibody at 4°C overnight and with secondary antibody at room temperature for 2 h. For staining G4, cells were treated with BG4 antibody at 4°C overnight, followed by anti-FLAG antibody, then anti-rabbit secondary antibody. For T-FISH, cells were dehydrated in 70, 95 and 100% ethanol. After air drying, TelC PNA probes (F1002-5; F1009-5, PANAGENE) were hybridized by boiling at 80°C for 15 min. For TERRA RNA-FISH, TelC PNA probes were hybridized at 37°C for 1 h. For telomere length measurements, cells were synchronized with 800 ng/ml Colcemid and incubated in 0.075 M KCl. Then, cells were fixed and dropped on Fisher-brand™ Superfrost Plus Microscope Slides (FIS#12–550-15; Fisher Scientific). Metaphase spreads were dehydrated and subjected to T-FISH as described above.