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In Situ Hybridization with Digoxigenin-labeled RNA probe (Paraffin Sections)

Dewaxing and Pretreatment of Slides

1. Warm slides at 55-60 °C for 1-1.5 hour.
2. Deparaffinize for 20min X 2 with 55-60 °C xylene.
3. Rehydrate:
   100% ETOH 2min X2
   95% ETOH 2min
4. Wash with 0.9% NaCl for 5min.
   5M NaCl 31.5mL to 1000ml DepH₂O
5. Incubate with 0.2M HCl for 15min at RT.
   3.4mL 12N HCl to 200ml H₂O
6. PBS 1min X 3.
7. Incubate with Proteinase K 20ug/mL in 50mM Tris pH 7.6, 5mM
   EDTA for 8min at RT.
   1M Tris 7.6 10ml
   0.5 M EDTA 2ml
   ddH₂O 188mL
   PK (10mg/ml) 400uL (aliquot in -20°C, to be added
   immediately before incubation.)
8. 0.4% Glycine in PBS for 2min X 2 at RT.
   Glycine 1.8g to 450ml PBS
9. 0.9% NaCl for 2min X 2.
10. 0.1M Triethanolamine-HCl pH 8.0 on magnetic stirrer.
    Stock: 1M Triethanolamine pH 8.0 (1000ml)
    Triethanolamine: 185.7g
    10N NaOH (20g NaOH in 50ml DepH₂O) 46-47ml
    
    Add DepH₂O to total volume 1000ml. Check pH
    
    Working Solution:
    0.1M Triethanolamine pH 8.0: (200ml)
    1M Triethanolamine 20mL
    ddH₂O 180mL
    
    Place RNAse free stir bar into staining dish. Place dish on stirrers.
    
    
11. Add 500uL of Acetic Anhydride to staining dish while stirring.
    Stir for 5min. Add again 500uL of Acetic Anhydride. Stir for 5min.
12. Rinse in Dep H₂O X2.
13. Dehydration:
    95% EtOH 1min
    100% EtOH 1minX2
14. Air dry in dust free area. Slides can then proceed to
15. hybridization step or be stored at -80 °C.

Hybridization

Hyb. Mix is aliquoted and stored at -80°C

1. Thaw Hyb. Mix.
2. Add Hyb. Mix to each new tube (60uL per slide)
3. Add probe to make final concentration
   1:10, 1:25, 1:50. 1:100
4. Mix probe by vortex and then spin down probe, keep mixed probe on ice.
5. Add 60uL probe to 24 X 40mm coverglass. Turn slides tissue side down and contact tissue to the probe. Turn slides back with coverglass at top.
6. Hybridize overnight at 55°C in humidity chamber ( 50% formamide and 2X SSC).

Posthybridization:

1. Warm 2X SSC at 60°C and STE at 37 °C.
2. Put slides into washing container with prewarmed 2XSSC.
3. Remove coverglass from slides. Chek no coverglass left in slides rack. Wash for 15min at 60°C.
4. Change prewarmed 2X SSC and wash for 30min at 60°C.
5. Wash with STE at 37°C for 20min.
   1XSTE: (1000ml)
   20X SSC 200ml
   1M Tris pH7.6 20ml
   0.5M EDTA 2ml
   ddH₂O 778ml
6. Wash with STE at 37°C for 10min.
7. Wash with 2X SSC for 30 min at 60°C.
8. Wash with 0.5X SSC for 30 min at 60°C.
9. Wash with 0.1X SSC for 30 min at 60°C.
10. Rinse with 0.1X SSC at RT.
11. ddH₂O at RT X2.

Immunological Detection

1. Wash with TBST 10minX3
2. Incubate with Tissue Blocking Solution for 30-60 min.
   2% Blocking Solution 8ml
   Normal Goat Serum 2ml
3. Incubate with anti-DIG Ab 1:1500 with Tissue Blocking Solution O/N at 4°C.
4. Wash with TBST 10minX6.
5. Change to NTMT with 2mM levamisole 5minX2.
   
   NTMT (Add fresh levamisole every time)
   
   Total Volume 50ml
   
   5M NaCl (100mM ) 1ml
   1M Tris-HCl pH 8,0 ( 100 mM ) 5ml
   1M MgCl2 ( 50mM ) 2.5ml
   Tween 20 (1% ) 0.5ml
   levamisole ( 2 mM ) 25mg
   ddH₂O
   
   
6. Incubate with Detection Solution:
   
   Total volume: 10ml
   
   NTMT 10ml
   NBT (Roche#1383213) 50 ul
   (final 0.5 mg/ml)
   BCIP (Roche#1383221) 38ul
   (final 0.1 mg/ml )
   
   
7. Develop and check under microscope.
8. Wash with ddH₂O and countstain with nuclear fast red for 10 min.
9. Wash with H₂O, dry at room temperature and mount.

 

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