4.13.3. Hybridization Analysis

  1. Pre-hybridize the sections in pre-hybridization solution (50% formamide, 5X SSC, 40 mg/ml salmon sperm) at 58oC for two hours.
  2. Incubate in hybridization buffer with Dig-labeled TARGET probe solution to a concentration of 100-200 ng/ml of probe in pre-hybridization solution at 58oC overnight.
  3. After hybridization, wash the sections twice in low stringency wash solution (2X SSC, 0.1% SDS) at room temperature for five minutes.
  4. Wash twice in high stringency wash solution (0.1 × SSC, 0.1 % SDS) at 52oC for 15 min.
    Note: The hybridization signals are detected with Alkaline phosphatase (AP)-labeled sheep anti-DIG antibody conjugate (Roche Applied Science).
  5. Add the conjugate solution to the dry sections.
  6. Incubate at 4°C for two hours in a chamber in which humidity is maintained at >70% relative humidity.
  7. Rinse the slides three times with washing buffers.
  8. Perform the colour development by adding the buffer solution containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) on the tissue sections.
  9. Incubate for three to six hours at RT protected from bright light.
  10. Stop the colour reaction by a 5 min wash in Tris/EDTA (0.1 mM, pH 8.0).
  11. Remove the non-specific staining in 95% EtOH overnight.
  12. Rehydrate the sections through:
    1. Successive incubation in ethanol (70%, 95%, and 100%).
    2. Incubation in xylol (2 X15 min each).
  13. Mount in Eukitt® resin (Sigma).
    Note: Negative control reactions include regular dUTP instead of DIG-labeled TARGET probe.
  14. Observe and photograph In Situ hybridization slides under a light microscope.

The hybridization signals are shown by dark blue sites where the DIG-labeled probe bound directly to the viral RNA. The section of negative control will stain pink only with the nuclear fast red.