Alexa 488 ccctaa 4 pna probe

After secondary-antibody incubation, cells were washed as above, but then the IF staining was fixed with 2% paraformaldehyde (PFA) for 10 min. PFA was washed off with PBS and coverslips dehydrated with successive washes in 70%, 95% and 100% ethanol for 3 min, and were allowed to air dry completely. Next, the coverslips were mounted on glass slides with 15 μl per coverslip of hybridization mix (70% deionized formamide, 1 mg ml⁻¹ of Blocking Reagent (Roche), 10 mM Tris-HCl pH 7.4) containing Alexa 488–(CCCTAA)₄ PNA probe (PNA Bio). DNA was denatured by setting the slides on a heating block set to 72 °C for 10 min and then incubating for at least 4 h or overnight at RT in the dark. The coverslips were then washed twice for 15 min with Wash Solution A (70% deionized formamide and 10 mM Tris-HCl pH 7.2) and 3 times with Solution B (0.1 M Tris-HCl pH7.2, 0.15 M NaCl and 0.08% Tween) for 5 min at RT. Ethanol dehydration was repeated as above, and finally the samples were mounted and analyzed as described above.