FISH

FISH was performed as recently reported [14]. A hybridization solution containing 50% deionized formamide, 10% dextran sulfate, 2×SSC (300 mM sodium chloride, 0.3 mM trisodium citrate), 0.33% SDS, 50 ng of the digoxigenated pTa71 probe and 2 pmol of the biotinated (AG)₁₀ probe was denatured in an oven for 10 min at 70°C and 30 μL, then used to flush the sample-bearing slides. The cells were then denatured by placing the slides in an incubator at 75°C for 7 min, with the temperature controlled using a programmable thermal controller (PT-100, M.J. Research). Hybridization was performed by incubating the slides overnight in a humidified chamber at 37°C. The slides were then washed for 10 min in 4× SSC/Tween²⁰. Digoxigenin and biotin were detected by incubating the slides in fluoresceinated anti-digoxigenin (Roche Applied Science) and streptavidin-Cy3 (Sigma-Aldrich), respectively, in 5% (w/v) bovine serum albumin for 1 h at 37°C. The slides were then rinsed for 10 min in 4× SSC/Tween²⁰ at RT, stained with the DNA stain DAPI (4´, 6-diamidino-2-phenylindole) and mounted in antifade solution (Vectashield, Vector Labs, Curlingame, CA).