(3:1 methanol:acetic acid) were dropped onto precleaned microscope slides, followed by fixation in acetone (Sigma–Aldrich) for 10 min and dehydration through an ethanol series (70%, 90% and 100%). Metaphase spreads on slides were denatured by immersion in an alkaline denaturation solution (0.5 M NaOH, 1.0 M NaCl) for 2 min, followed by rinsing in 1 M Tris-HCl (pH 7.4) solution for 3 min, 1× PBS for 3 min and dehydration through a 70%, 90% and 100% ethanol series. The M-FISH probes were denatured at 65 °C for 10 min before being applied onto the denatured slides. The hybridization area was sealed with a 22 mm × 22 mm coverslip and rubber cement. Hybridization was carried out in a 37 °C incubator for approximately 44–48 h. The post-hybridization washes included a 5-min stringent wash in 0.5× SSC at 75 °C, followed by a 5 min rinse in 2× SSC containing 0.05% Tween-20 (VWR) and a 2 min rinse in 1× PBS, both at room temperature. Finally, slides were mounted with SlowFade Gold mounting solution containing DAPI (Thermo Fisher Scientific). M-FISH images were visualized on a Zeiss AxioImager D1 fluorescent microscope equipped with narrow band-pass filters for DAPI, DEAC, FITC, Cy3, Texas
