Two Day In Situ Hybridization Protocol for Paraffin Sections

In situ hybridisation is performed using the high-throughput robot manufactured by Tecan.

Day 1

  1. Deparaffinize sections 2x10 minutes in xylene
  2. Rehydrate thru ethanol series 2 minutes in each of 100, 95, 70, 50, and 1x Fish Saline.
  3. Incubate in 2.5-ug/ml proteinase K (20 mg/ml stock) in 1x Fish Saline 30 minutes 37°C.
  4. Wash 2-mg/ml glycine in 1x Fish Saline Room Temperature
  5. Wash in 0.1 M triethanolamine pH 7.5 for 5 minutes Room Temperature.
  6. Wash in 0.1 M triethanolamine pH 7.5 for 10 minutes at Room Temperature following the addition of 125 mls of acetic anhydride.
  7. Rinse in 2x SSC 2 minutes Room Temperature
  8. Dehydrate thru ethanol series 2 minutes in each of 50, 95 and 100
  9. Air dry at least 2 hours RNase Free
  10. Place 50 µl of digoxigenin labeled riboprobe on each slide and coverslip (coverslips should be from unopened box; these should be then reserved for hybridizations only).
  11. Incubate overnight at 47-50 °C in humid chamber (glass baking dish (RNase free) lined with blotting paper or filter paper soaked with 50% formamide in 2 x SSC).

Day 2

  1. Remove cover slips gently in 2x SSC
  2. Incubate 1 hour in 50% formamide in 2x SSC at 47-50 °C
  3. Rinse in 10mM Tris pH 8.0 (1M stock); 500 mM sodium chloride (5M stock) for 2 minutes at Room Temperature
  4. Incubate in the same buffer with 100 ul RNase A (10 mg/ml stock) and 5 µl RNase T1 (100 000 Units) at 37 °C for 30 minutes
  5. Rinse in same buffer without RNase 30 minutes at 37 °C
  6. Wash 30 minutes in each of 2x SSC, 0.5x SSC, and 0.1x SSC at 47-50 °C
  7. Remove from last wash and Rinse in 0.1 M Tris pH 7.5 (1M stock); 150 mM NaCl for 2 minutes
  8. Incubate in same buffer with 1% BSA and 10% goat serum for 30 minutes at Room Temperature Make up 1/250 dilution of alkaline phosphatase anti-digoxigenin antibody in the same buffer.
  9. Incubate at room temperature for 30 minutes in a humid chamber (glass baking dish lined with filter paper soaked with the same buffer). Place 500 ul of antibody solution on each slide. Do not add cover slip.
  10. Wash in the same buffer w/o goat serum but with Tween 20 for 30 minutes at Room Temperature with gentle rocking
  11. Wash 15 minutes in chromogenic buffer plus 9.68 mg levamisol Chromogenic buffer: 4.00 ml 1M Tris pH 9.5 1.00 ml 1M MgCl2 0.80 ml 5M NaCl 0.04 ml Tween-20 Make up to 40 ml with DW
  12. Make up chromogenic solution: Add 0.0045 ml NBT 0.0035 mls BCIP per ml of chromogenic buffer Incubate slides in solution until good signal is achieved (around 20 minutes)
  13. Stop reaction in 1x Fish Saline
  14. Rapidly dehydrate through ethanol series 50, 95, 100 and clear in xylene.
  15. Mount in cytoseal and dry overnight Fish Saline (10x) pH 7.8 350.0 ml, 5 M NaCl 27.0 ml, 1 M KCl 27.4 ml, 1 M CaCl2.H2O 6.4 ml, 1 M MgCl2.H2O 22.2 ml, 1 M Glucose 30.0 ml, 1 M Tris Base 463 mls, + 537 mls DW. Autoclave.