3.4.1. Hybridization procedures

  1. 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.
  2. 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.
  3. Apply 150 µl of the hybridization buffer onto the sections and cover with a coverslip, taking care not to trap air bubbles.
  4. Put the glass slides in a humidified chamber and leave at room temperature, overnight, in the dark.
  5. 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.
  6. 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.
  7. 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.
  8. Seal all sides of the coverslip with clear fingernail polish.
  9. 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.