Denaturation and Hybridization
Fifteen microliters of hybridization mixture consisting of 0.3 µg/mL Cy3-labeled, telomere-specific PNA probe; 0.1 µg/mL FITC-labeled, chromosome 9-specific Sat III PNA probe; 50% formamide; 10 mM Tris-HCl, pH 7.5; 5% blocking reagent; and 1X Denhart’s solution was applied to each slide and covered under a cover slip.
Before starting the procedure, SciGene’s Hybex microarray incubation system was preheated to 75°C with a water bath insert in position. Fifteen microliters of hybridization mixture was pipetted onto each slide, and the spread area was covered with a cover slip, avoiding the creation of air bubbles.
Then, the slides were inserted into the Hybex slide racks, which were mounted on a hybridization chamber base (Figure 1A). One milliliter of water was pipetted on the absorbent pad situated in the chamber cover to provide adequate moisture during hybridization. The cover of the chamber was sealed to the base by tightening the corner screws (Figure 1B), and a black handle attached. The water bath insert was removed from the preheated Hybex unit, and the assembled chamber was placed into the heating unit, using both chambers to ensure accurate heat control (Figure 1C).
Once the system temperature equilibrated to 75°C (in 8–10 minutes), the slides were incubated for five minutes. Afterward, the hybridization chambers were removed and left at room temperature for 15 minutes. The Hybex unit was speed-cooled by setting the temperature to 30°C and placing a water bath insert into the unit. When 30°C was reached, the hybridization chambers were placed back into the instrument and incubated for three hours. (For DNA probes, hybridization can be performed at 37–42°C for 10 to 16 hours.)