Telomere and γH2AX Immuno-FISH Protocol

Cells were collected by shake-off after double thymidine synchronization to G₂/M phase, and swollen in 0.075 M KCl hypotonic buffer for 10 min at 37 °C. The cells were fixed by 1% formaldehyde in PBS for 2 min, and then spun onto coverslips using a cytospin apparatus (Cytospin). Chromosome spreads were fixed again in 4% formaldehyde in PBS for 15 min, followed by permeabilization in 0.5% Triton X-100/PBS for 15 min at room temperature. For the telomere PNA-γH2AX immuno-FISH^{34 (link)}, the mitotic cells on slides were incubated with TAMRA-OO-[CCCTAA]3 labeled PNA probe (PANAGENE, Cat. no. F2001) at 85 °C for 2 min, then incubated in 37 °C overnight. After formamide fixation, the cells were stained with γH2AX antibody. DAPI counter staining was performed to label the nuclei or chromosome. Stained slides were analyzed using a Leica TCS SP5 II scanning confocal microscope.