RNA FISH

1. Cells are seeded into uncoated glass culture slides (BD Biosciences) and incubated for 18 hours.
2. The cells are then fixed with 4% formaldehyde for 30 mins at room temperature, washed with PBS and permeabilized in 70% ethanol at 4 °C over night.
3. The cells are rehydrated and washed with PBS (3x5mins).
4. The cells are incubated in a freshly prepared solution (0.13M 1-methylimidazole, 300mM NaCl, pH 8.0) (2x10mins), followed by incubation in solution (0.16M l-ethyl-3-(3–dimethylaminopropyl) carbodiimide (EDC) (Sigma) for 1 h at room temperature.
5. The cells are washed in 0.2% (w/v) glycine/TBS (25mM Tris Ph 7.4, 150mM NaCl, 2.5mM KCl, 0.1% Triton-X100), and then washed in TBS twice.
6. The cells are then incubated in prehybridization buffer (25% formamide, 0.05M EDTA, 4xSSC, 10% dextran sulfate, 1x Denhardt’s solution, 0.5 mg/ml Escherichia coli tRNA and 0.5 mg/ml RVC) for 2 h at 60 °C .
7. The cells are then incubated in hybridization buffer (prehybridization buffer including 10 nM LNA probe (Exiqon, digoxigenin was labeled at the 3 end of the probe) for 1 h at 60 °C .
8. Wash the cells in 4xSSC, 2x SSC and 1xSSC (10min/each buffer at 65℃ ).
9. Incubate the cells in the blocking buffer (10% normal goat serum in PBS) for 1 h at room temperature.
10. Incubate the cells with anti-DIG antibody (1:400, Roche) in the cold room overnight.
11. After three washes in PBS, Rhodamine Red labeled secondary antibody (1:400, Invitrogen) was added and incubated at room temperature for 1 h.
12. The slides were then counterstained in DAPI (Invitrogen) and mounted in mounting solution.

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