Ttaggg 3 alexa488

Sub-confluent ESCs were incubated with 5’-bromo-2’-deoxyuridine (BrdU; Sigma) at the concentration of 10 mM for 12–14 h. Colcemide was added at a concentration of 0.1 μg/ml during the last 3–4 hours, and metaphase spreads were prepared for telomere Q-FISH. Chromosome slides were treated with RNaseA, fixed with 4% formaldehyde, then stained with Hoechst 33,258 (0.5 mg/ml) for 15 min and exposed to 365 nm UV light for 40 min. The BrdU-substituted DNA was digested with Exonuclease III (Takara). PNA-FISH was performed with telomeric C-rich probe (CCCTAA)₃-Cy3 (Panagene, F1002) and G-rich probe (TTAGGG)₃-Alexa488 (Panagene, F1008). Images were captured using microscope Leica DM6B using a 100× oil objective.

Sub-confluent ESCs were incubated with 5’-bromo-2’-deoxyuridine (BrdU; Sigma) at the concentration of 10 mM for 12–14 h. Colcemide was added at a concentration of 0.1 μg/ml during the last 3–4 hours, and metaphase spreads were prepared for telomere Q-FISH. Chromosome slides were treated with RNaseA, fixed with 4% formaldehyde, then stained with Hoechst 33,258 (0.5 mg/ml) for 15 min and exposed to 365 nm UV light for 40 min. The BrdU-substituted DNA was digested with Exonuclease III (Takara). PNA-FISH was performed with telomeric C-rich probe (CCCTAA)₃-Cy3 (Panagene, F1002) and G-rich probe (TTAGGG)₃-Alexa488 (Panagene, F1008). Images were captured using microscope Leica DM6B using a 100× oil objective.