Telomere FISH

Cells were incubated for 5 h in 0.1 μg/mL KaryoMAX colcemid (Gibco 15212–012), collected and incubated in 75 mM KCl for 30 minutes, then fixed three times in methanol:acetic acid (3:1). Following fixation, cells were dropped onto microscope slides and metaphase spreads were allowed to dry overnight then rehydrated in 1x PBS, followed by fixation in 4% paraformaldehyde for 5 min. Slides were then washed in 1x PBS, dehydrated in an ethanol series (70%, 95%, 100%) pre-chilled to −20°C and air dried. FISH was performed with TelC-Alexa488 (PNA Bio F1004) in hybridization buffer [70% formamide, 10 mM Tris pH 7.2, 0.5% including blocking buffer pH 7.5 (100 mM maleic acid, 150 mM NaCl, 10% Roche blocking reagent #11096176001] preheated to 80°C. Slides were denatured with the probe at 80°C, then allowed to incubate overnight at RT in a humid chamber. Next, slides were washed twice in hybridization wash A (10 mM Tris-HCl pH 7.2, 0.1% BSA, 70% formamide) and three times in hybridization wash B (0.1 M Tris-HCl pH 7.2, 0.15 M NaCl, 0.08% Tween-20). The second hybridization wash B contained DAPI (Life Technologies D1306) at a 1:1000 concentration. Slides were then dehydrated in an ethanol series (70%, 95%, 100%) pre-chilled to −20°C, air dried and mounted using Prolong gold anti-fade mountant (Invitrogen {"type":"entrez-protein","attrs":{"text":"P36930","term_id":"1248281091","term_text":"P36930"}}P36930). Images were captured using NIS element software with a Nikon i90 microscope. Images were scored manually. Statistically analysis was performed using GraphPad Prism 9.