1. Prehybridization buffer: 5x SSC, 0.1% SDS and 1% BSA (Sigma #B-4287). Heat to 50ºC while stirring.
2. Slides to be analyzed are placed in a staining jar; prehybridization buffer is added, and incubation is carried out at 48ºC for 45-60 min while stirring.
3. The slides are washed by dipping up and down approximately 10 times in two different staining jars of deionized water. Excess water is removed by shaking the slide rack up and down two times.
4. The slides are then dipped in an up and down motion approximately 10 times at room-temperature in isopropanol and spun dried. The slides are used immediately after prehybridization (less than 1 hr) as hybridization efficiency decreases rapidly if the slides are allowed to dry for more than that time. Hybridization:
5. 2X hybridization buffer: 50% formamide, 10X SSC and 0.2% SDS. Incubate the solution until it reaches 48°C.
6. The Cy3 / Cy5 labeled mixtures are re-suspended in 9 µl water, and heated to 95°C for 3 min to denature, and are centrifuged at maximum angular velocity for 1 min.
7. The following are added to each tube in order to block non-specific hybridization. Make a master-mix with the following ingredients for each tube: • Calf Thymus DNA (1µg/µL) 8µl (Sigma; Cat #D 8661) • poly(A)-DNA (10mg/mL) 2µl (Sigma #P 9403) • yeast tRNA (4mg/mL) 2µl (Sigma #R 8759)
8. Then, 21 µl 2X hybridization buffer that has been pre-heated to 48°C is added to the target mixture, mixed well, and centrifuged. The samples are kept at 48°C until placed on the slide.
9. The labeled target is applied to a pre-hybridized microarray slide and covered with a 22 x 60 mm glass cover slip.
10. The slide is placed in a sealed hybridization chamber (Corning. Acton, MA), and 12 µl water is added to the small reservoirs at each end of the chamber.
11. The sealed chamber is placed in a 48°C water bath and incubated for 40-60hr (2)
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