Zytolight spec cdk4 cen12 dual color probe

Slides were fixed in MeOH:acetic acid for 10 min at −20 °C followed by a wash of the slide in PBS for 5 min at room temperature. Slides were incubated in pepsin solution (0.001 N HCl) with the addition of 10 µl pepsin (1 g 50 ml⁻¹) at 37 °C for 10 min. Slides were washed in 0.5× saline-sodium citrate (SSC) buffer for 5 min and dehydrated by washing in 70%, 90% and 100% cold ethanol (stored at −20 °C) for 3 min. Dried slides were stained with 10 µl Vysis LSI N-MYC SpectrumGreen/CEP 2 SpectrumOrange Probes (Abbott), ZytoLight SPEC CDK4/CEN 12 Dual Color Probe (ZytoVision) or ZytoLight SPEC MDM2/CEN 12 Dual Color Probe (ZytoVision), covered with a coverslip and sealed with rubber cement. Denaturing occurred in a ThermoBrite system (Abbott) for 5 min at 72 °C followed by 37 °C overnight incubation. The slides were washed for 5 min at room temperature in 2× SSC/0.1% IGEPAL, followed by 3 min at 60 °C in 0.4× SSC/0.3% IGEPAL (Sigma-Aldrich) and an additional wash in 2× SSC/0.1% IGEPAL for 3 min at room temperature. Dried slides were stained with 12 µl Hoechst 33342 (10 µM, Thermo Fisher Scientific) for 10 min and washed with PBS for 5 min. After drying, a coverslip was mounted on the slide and sealed with nail polish. Images were taken using a Leica SP5 Confocal microscope (Leica Microsystems).