Axio imager m1 microscope

Telomeric Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH) was performed as described previously (Lansdorp et al., 1996 (link)). Briefly, cells were treated with 0.2 μg/ml of colcemid (Roche) for 90 minutes to arrest cells in metaphase. Trypsinized cells were incubated in 75 mM KCl and fixed with methanol:acetic acid (3:1 ratio). The cells were then dropped onto glass slides and left to dry for at least 24 hours. The slides were rehydrated in PBS for 5 minutes, fixed in 4% formaldehyde for 5 minutes, treated with 1 mg/ml of pepsin for 10 minutes at 37°C, and fixed again in 4% formaldehyde for 5 minutes. Next, slides were dehydrated in 70%, 85%, and 100% (v/v) ethanol for 15 minutes each and then air-dried. Metaphase chromosome spreads were hybridized with a telomeric FITC-TelC PNA probe in hybridization buffer (70% formamide, 0.5% blocking reagent (Roche), 10 mM Tris-HCl pH 7.2) for 1 hour at room temperature, followed by 2 washes in wash buffer (70% formamide, 10mM Tris-HCl pH 7.2) for 15 min each and 3 washes in 1X PBS for 5 min each. The slides were mounted using ProLong Gold antifade with DAPI (Thermo Fisher). Chromosome images and telomere signals were captured using Zeiss Axio Imager M1 microscope equipped with an ORCA-ER camera (Hamamatsu) controlled by Volocity 6.3 software (Improvision).