1. Keep the Hybridization Buffer containing the probe. It can be reused up to two times. Replace hybridization buffer with a solution of 50% formamide, 5X SSC (Mock-Hyb Buffer). Hybridization buffer can be used here, but it is more economical to use the Mock-Hyb Buffer. Wash at 60°C for 10 min.

2. Wash in 50% Mock-Hyb buffer/50% 2X SSC 10 min at 60°C.

3. Wash in 25% Mock-Hyb buffer/75% 2X SSC for 10 min at 60°C.

4. Wash twice for 20 min in 2X SSC/0.3% CHAPS at 60°C.

5. RNAse treat by incubating for 30 min at 37°C in 2X SSC/0.3% CHAPS containing 20 |g/mL RNase A and 10 U/mL RNase T^

6. Rinse in 2X SSC/0.3% CHAPS for 10 min at room temperature.

7. Wash twice for 30 min in 0.2X SSC/0.3% CHAPS at 60°C.

8. Wash twice for 10 min in PBST/0.3% CHAPS at 60°C.

9. Wash three times for 5 min in PBST at room temperature.

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