3.4.1. Hybridization procedures
- Freshly prepare a hybridization buffer containing the probe(s) (20 mM Tris-HCl [pH 8.0], 0.9M NaCl, 0.01% sodium dodecyl sulphate, 30% formamide, 100 pmol/ml each of the probes, 10 µg/ml DAPI or equivalents). Prepare 150 µl of this solution per slide.
- Remove paraffin from the sections using absolute xylene (soak for 3 min., and repeat this 3 times), followed by absolute ethanol (soak for 3 min, twice), then rinse with RNase-free water.
- Apply 150 µl of the hybridization buffer onto the sections and cover with a coverslip, taking care not to trap air bubbles.
- Put the glass slides in a humidified chamber and leave at room temperature, overnight, in the dark.
- The next day, carefully remove the coverslip by applying freshly prepared PBSTx (0.8% NaCl [w/v], 0.02% KCl [w/v], 0.115% Na2HPO4 [w/v], 0.02% KH2PO4 [w/v], 0.3% Triton X-100 [v/v]) between the glass slide and coverslip using a wash bottle.
- Wash the slides with PBSTx with gentle agitation (~50 rounds per minute) in the dark, 3 times for 10 min at room temperature. You can use conventional staining trays or containers.
- Apply 100 µl of a commercially available antifade solution such as SlowFade (Invitrogen) or DABCO-glycerol (prepared by mixing 1.25 g of 1,4-Diazabicyclo[2.2.2]octane [DABCO], 10 mL of PBS and 90 mL of glycerol) directly onto the specimen and cover the slide with a coverslip; avoid trapping air bubbles.
- Seal all sides of the coverslip with clear fingernail polish.
- The samples are now ready for microscopy and should be immediately analysed. These slides cannot be stored because the rRNA probes and fluorochromes are unstable, especially in the light.