For all procedures after hybridization, we use separate glassware and graded ethanol solutions from those used prior to hybridization because RNase A is used during the washing procedures and will potentially contaminate all glassware and reused solutions.
1. Place slides in 4X SSC at room temperature until cover slips fall off (about 20 min).
2. Remove cover slips, and place slides in fresh 4X SSC for 1 h.
3. Meanwhile, make up 1.6 L of TNE buffer and 80 mL of high-stringency buffer/ 25 slides, prewarming the latter to 50°C or 65°C as appropriate (see step 4).
4. Place slides back to back in Coplin jars containing high-stringency buffer in a 65°C water bath (high stringency) or 50°C (low stringency) for 30 min (see Note 4).
5. Transfer slides to a rack, and place in 350 mL of TNE buffer for 10 min at 37°C. Then repeat this wash once more.
6. Place slides in a third trough containing 400 mL prewarmed TNE buffer and 800 pL of 10 mg/mL RNase A for 30 min at 37°C.
7. Wash slides for 10 min in fresh TNE buffer at 37°C.
8. Wash slides for 30 min in Coplin jars containing prewarmed high-stringency buffer at 65°C (or appropriate temperature).
9. Wash for 15 min in 2X SSC, and then for 15 min in 0.1X SSC both at room temperature.
10. Dehydrate through graded ethanols containing 2.5 M ammonium acetate (the latter to prevent "melting" of the probe from target RNAs) from 30 to 100% by dipping as before.
11. Air-dry in a rack loosely wrapped in foil for 60 min minimum. The slides may be left overnight to dry.
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