In Situ Hybridization Protocol Using DIG-Labeled cRNA Probe

 

  1. Fix cryo-sections (10 mm) in ice-cool 4% paraformaldehyde for 15 mins.
  2. Wash with  PBS (made in DEPC water) for 15x2 min at room temperature.
  3. Prehybridize for 2 h at 55°C. Prehybridization solution: 50% deionized formamide, 5xSSC, 120 mg/ml salmon sperm DNA, 10% dextran sulfate, 1xDenhardt’s reagent.
  4. Hybridize using DIG-labeled cRNA probe overnight at 55°C. Use the prehybridization solution to dilute probe. (Probe conc: 400 ng/ml, for practical purposes, you may use the probe generated by one DIG labeling reaction for as many as 8 slides; denature the probe at 70°C for 5 min and quickly chill on ice prior to adding to the slides. Make DIG labeled riboprobe using manufacturer’s instructions supplied by Roche Molecular Biochemicals).
  5. Wash slides 30 min with 2xSSC at room temperature.
  6. Digest with RNase A (100ng/ml in 50 mM Tris and 150 mM NaCl, pH=7.4) at 37°C for for 20 min.
  7. Wash slides 30 min with 2xSSC at room temperature.
  8. Wash slides 1 h with 2xSSC at 68°C.
  9. Wash slides 1 h with 0.1xSSC at 68°C.
  10. Incubate slides with blocking solution for 30 min at RT. Blocking reagent: bought from Roche Molecular Biochemicals, Cat#1096176 and it was dissolved in maleic acid buffer according to manufacturer’s instruction.
  11. Incubate slides with anti DIG-AP fragment (1:2000) in blocking solution for 2 h at room temperature.
  12. Wash slides with 50 mM Tris/150 mM NaCl (pH=7.4) for 15x2 min at room temperature.
  13. Incubate slides in 0.1M Tris/0.1M NaCl/0.05 M MgCl2 (pH=9.5) for 5 min at room temperature.
  14. Develop color using NBT-BCIP (Bought from Roche Molecular Biochemicals, Cat # 1681451; 20 ml/ml of buffer in step#13). Color development takes 2-8h.
  15. Wash slides extensively with water and mount coverslip (Mounting medium: Same as used for immunocytochemistry).