Prehybridization Treatment and Hybridization

1. Dip slides in Histolene two times for 10 min to remove wax (see Note 4).

2. Rehydrate through graded alcohols (1 min in each): 100% MeOH (twice), 75% MeOH/25% PBT, 50% Me0H/50% PBT, 25% MeOH/75% PBT.

3. Immerse slides in PBT for 5 min.

4. Overlay the slides with Proteinase K, (10 |g/mL, prewarmed to 37°C) and leave for 15 min at room temperature.

5. Wash three times for 1 min with PBT.

6. Immerse the slides in ice-cold 4% cold paraformaldehyde in PBS and leave for 20 min at room temperature.

7. Wash the slides with PBT, twice for 1 min.

8. Add probe to hybridization solution (10-200 ng/slide—actual amount determined empirically). Heat to 80°C for 5 min to ensure uniform solution and quench in ice.

9. Apply the hybridization mix (approx 70 | L/slide) to the sections and cover with a coverslip. Incubate overnight at 40-70°C in a box humifidified with 50% formamide, 5X SSC (we routinely use 70°C for intact riboprobes 1-1.5 kb) (see Note 5).

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