In Situ Hybridization with Digoxigenin-labeled RNA probe (Paraffin Sections)
Dewaxing and Pretreatment of Slides
- Warm slides at 55-60 °C for 1-1.5 hour.
- Deparaffinize for 20min X 2 with 55-60 °C xylene.
100% ETOH 2min X2
95% ETOH 2min
- Wash with 0.9% NaCl for 5min.
5M NaCl 31.5mL to 1000ml DepH2O
- Incubate with 0.2M HCl for 15min at RT.
3.4mL 12N HCl to 200ml H2O
- PBS 1min X 3.
- 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
PK (10mg/ml) 400uL (aliquot in -20°C, to be added
immediately before incubation.)
- 0.4% Glycine in PBS for 2min X 2 at RT.
Glycine 1.8g to 450ml PBS
- 0.9% NaCl for 2min X 2.
- 0.1M Triethanolamine-HCl pH 8.0 on magnetic stirrer.
Stock: 1M Triethanolamine pH 8.0 (1000ml)
10N NaOH (20g NaOH in 50ml DepH2O) 46-47ml
Add DepH2O to total volume 1000ml. Check pH
0.1M Triethanolamine pH 8.0: (200ml)
1M Triethanolamine 20mL
Place RNAse free stir bar into staining dish. Place dish on stirrers.
- Add 500uL of Acetic Anhydride to staining dish while stirring.
Stir for 5min. Add again 500uL of Acetic Anhydride. Stir for 5min.
- Rinse in Dep H2O X2.
95% EtOH 1min
100% EtOH 1minX2
- Air dry in dust free area. Slides can then proceed to
- hybridization step or be stored at -80 °C.
Hyb. Mix is aliquoted and stored at -80°C
- Thaw Hyb. Mix.
- Add Hyb. Mix to each new tube (60uL per slide)
- Add probe to make final concentration
1:10, 1:25, 1:50. 1:100
- Mix probe by vortex and then spin down probe, keep mixed probe on ice.
- 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.
- Hybridize overnight at 55°C in humidity chamber ( 50% formamide and 2X SSC).
- Warm 2X SSC at 60°C and STE at 37 °C.
- Put slides into washing container with prewarmed 2XSSC.
- Remove coverglass from slides. Chek no coverglass left in slides rack. Wash for 15min at 60°C.
- Change prewarmed 2X SSC and wash for 30min at 60°C.
- Wash with STE at 37°C for 20min.
20X SSC 200ml
1M Tris pH7.6 20ml
0.5M EDTA 2ml
- Wash with STE at 37°C for 10min.
- Wash with 2X SSC for 30 min at 60°C.
- Wash with 0.5X SSC for 30 min at 60°C.
- Wash with 0.1X SSC for 30 min at 60°C.
- Rinse with 0.1X SSC at RT.
- ddH2O at RT X2.
- Wash with TBST 10minX3
- Incubate with Tissue Blocking Solution for 30-60 min.
2% Blocking Solution 8ml
Normal Goat Serum 2ml
- Incubate with anti-DIG Ab 1:1500 with Tissue Blocking Solution O/N at 4°C.
- Wash with TBST 10minX6.
- 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
- Incubate with Detection Solution:
Total volume: 10ml
NBT (Roche#1383213) 50 ul
(final 0.5 mg/ml)
BCIP (Roche#1383221) 38ul
(final 0.1 mg/ml )
- Develop and check under microscope.
- Wash with ddH2O and countstain with nuclear fast red for 10 min.
- Wash with H2O, dry at room temperature and mount.