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Protocol
miRNA and mRNA in situ hybridization
Keywords: In situ, ISH, hybridization, in situ hybridization, prostate, bladder, FFPE
Protocol for FFPE Bladder and Prostata tissue
Nitril gloves should always be worn when working with formamide. Work in biosafety cabinet and use sealed zip lock bags whenever you need to move your samples.
Instructions
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Solutions and buffers for ISH (1VGYAYS)
Nitril gloves should always be worn when working with formamide. Work in biosafety cabinet and use sealed zip lock bags whenever you need to move your samples.
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Proteinase K Stock Solution (5 mg/ml)
Dissolve 5 mg /ml of Proteinase K in Molecular grade water. Stored at -20˚ C.
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Levamisole Solution
Add levamisole to the NBT/BCIP to a final concentration of 2 mM. 0.258 g Levimisole Adjust to 10.75ml MilliQ H20
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NBT/BCIP and Levamisole Solution
Add levamisole to the NBT/BCIP to a final concentration of 2 mM.
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KTBTw stop solution (1000 ml)
50 ml Tris-HCl (pH 7.5 )(50 mM) 0.75 g KCl(10mM) 10 ml 100% Tween 20 (1%) 8.8 g NaCl (150 mM) Adjust to 1000 ml with MilliQ H20
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1 M Tris pH 7.5 (1000 ml)
Add 121 g Tris-Base to 700 ml H20 Adjust pH to 7.5 with concentrated HCL Adjust to 1000 ml with MilliQ H20
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1 M Tris pH 9.5 (1000 ml)
Add 121 g Tris-Base to 700 ml H20 Adjust pH to 9.5 with concentrated HCL Adjust to 1000 ml with MilliQ H20
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0.5 M Tris pH 8.0 (250 ml)
Add 15.1 g Tris-Base to 175 ml milliQ H20 Adjust pH to 8.0 with concentrated HCL Adjust to 250 ml with milliQ H20
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Yeast tRNA (25 mg/ml)
Dissolve 250 mg yeast tRNA in 10 ml of DEPC (Molecular grade water).
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Heparin 50 mg/ml
Dissolve 50 mg Heparin in 1 ml DEPC H20.
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Troubleshooting
Be careful when using PBS together with a AP based detection system as phosphate ions can inhibit the activity of the enzyme.
For DIG System applications do not include MgCl¬2 in the detection buffer as this might lead to spotty background on the slide after the detection procedure.
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Proteinase K Stock Solution (5 mg/ml)
Dissolve 5 mg /ml of Proteinase K in Molecular grade water. Stored at -20˚ C.
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50 mM Tris pH 7.5 (50 ml)
Dilute 2.5 ml of 1M Tris pH 7.5 in 47.5 ml H2O in a 50 ml Nunc tube.
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Proteinase K Working Solution (50 µg/ml)
Dilute the Proteinase stock solution 1µl Proteinase-K + 99 µl 50 mM Tris pH 7.5
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Acetic anhydride/triethanolamine
2.33 ml triethanolamine, 500 µl acetic anhydride, add to 200 ml with DEPC (Molecular grade water).
Use immediately!
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4% formaldehyde (200 ml)
22 ml 3 % formaldehyde (4%) Adjust to 200 ml with PBS DEPC (Molecular grade water).
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Prehybridization Buffer (50 ml)
25.0 ml 99,9% formamid,(50%) 50 µl 50 mg/ml heparin (50 µg/ml) 100 µl 25 µg/ml yeast RNA (50 µg/ml 1.0 g Blocking Powder (2%) 50 µl 100 % tween 20 (0,1%) 12.5 ml 20xSSC (5x) 0.093 g EDTA (5 mM) Adjust to 50 ml with DEPC H20
Store as aliquots (1 ml) at minus 80° C
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Hybridization Buffer
Molecular mass NaCl 58.44 g/mol
25.0 ml 99.9% formamid,(50%) 0.9 g Nacl (0.3M) 2.0 ml 0.5 M Tris-HCl pH 8.0 (20 mM) 5 g Dextran Sulphate (10%) 1 ml 50x Denhardts (1x) 1 ml 25 mg/ml yeast tRNA (0,5 mg/ml)Store as aliquots (1 ml) at minus 80° C
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Chamber Humidity Solution (500 ml)
250 ml 99.9% formamide (50%) 25 ml 20x SSC (1x) Adjust to 500 ml with MilliQ H20
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Washing buffer 1 (1000 ml)
500 ml 99,9% formamide (50%) 1,0 ml 100% Tween 20 (0,1%) 200 ml 5x SSC (1x) Adjust to 1000 ml with H20
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Washing buffer 2 (1000 ml)
10 ml 20x SSC (0,2x) Adjust to 1000 ml with H20
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Washing buffer 3 (1000 ml)
1 ml 100% Tween 20 (0.1% final) Adjust to 1000 ml with PBS
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Blocking Solution 50 ml
0,25 g blocking powder(0,5%) 5 ml heatinactivated goat serum (10%) 50 µl 100% Tween 20(0.1%) Adjust to 50 ml with PBS
Store aliquots (1000ul) at -20° C
Heat blocking powder at 50° C for a few hours or O/N
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Blocking Solution 50m to be stored at 5˚ degrees
0,25g blocking powder (0.5%) Adjust to 50 ml with PBS
Heat blocking powder at 50° C for a few hours or O/N
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Antibodies
1:250 or 1:500 anti DIG-AP Fab fragments (Roche 109327) in 0.5 % blocking buffer stored at 5˚ C.
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NTMTw (1000ml)
Molecular mass NaCl 58.44 g/mol; Molecular mass MgCl2 95.12 g/mol
5.8 g NaCl (Final conc. 100 mM) 100 ml 1 M stock Tris, pH 9.5 (Final conc. 100 mM) 4.7 g MgCl2 (Final conc. 50 mM) 1.0 ml 100% Tween 20 (Final conc. 0.1%) Adjust to 1000 ml with MilliQ H20Filter the solution with 0.45 µm filter to avoid MgCl2 precipitation.
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Deparaffinization
Ultraclear 10 min Ethanol 100% 10 min Ethanol 70% 5 min Molecular grade water 2 min
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Deproteination
- Wash for 15 min in PBS RNase free water (Molecular grade water)
- Digest with 200 µl of proteinase K (50 µg/ml) for 30 min at room temperature (RT) or if in a hurry for 15 min at 37˚ C.
- Adjust oven to hybridization temperature. For miR320 = 57.5° C
- Wash for 15 min in PBS RNase free water (Molecular grade water)
- Fix sections for 10 min in 4% formaldehyde and PBS (fresh made) at 4° C
- Wash for 15 min in PBS RNase free water (Molecular grade water)
- Treat with acetic anhydride/triethanolamine 5 min, mix and use solution immediately. 10 min at RT. (To block the unspecific background).
- Wash for 5 min in PBS RNase free water (Molecular grade water)
After this step continue in biosafety hood and remember to use nitril gloves when handling formamide.
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Prehybridization
Prehybridize slides for 3 hours and 15 min in prehybridization buffer (200 µl / slide) at hybridization temp. Hybridize slides covered with cover glass from Sigma in a humidified chamber with soaked wipes with 50% formamide and 1x SSC placed at the bottom of the chamber. Hybridization temperature should be 20-22° C lower than Tm (melting temperature).
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Hybridization
- Dilute probe to 5 pmol probe/µl (1 µl probe to 4 µl H20 RNase free water).
- Warm the diluted probe at 80° C for 5 min to linearize probe and chill on ice.
- Mix 1,5-2 µl diluted probe with 200 µl hybridization buffer (10 pmol) / slide
- Add 200 µl hybridization/probe mix per slide
- Hybridize slides overnight with cover glass in a humidified chamber- with soaked wipes with 50% formamide and 1x SSC placed at the bottom of chamber. Seal in a zip lock bag. Hybridization temperature should be 20-22° C lower than Tm (melting temperature).
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Stringency Wash
- Rinse in 5x SSC for 15-20 min with gentle agitation to remove cover glass.
- Wash for 60 min in washing buffer 1 at hybridization temperature - if signal is too weak reduce temperature, if too much background increase temperature.
- Wash for 15 min in washing buffer 2 at RT.
- Wash for 15 min in PBS at RT.
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Immunological Detection
- Block for 1 hour in 200 µl blocking solution (stored at -20° C)
- Incubate for 2 hours with antibody 1:250 anti DIG at RT in blocking buffer. (Stored at 5° C). Use between 200-300 µl depending on the size of the sample.
- Wash 2x30 min in washing buffer 3
- Wash in 2x20 min in PBS
- Wash in NTMTw for 5 min
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Color Reaction
- Incubate in 250 µl NBT/BCIP and Levamisole per slide in the dark at RT O/N
- Wash in KTBTw stop solution for 5 min. Wash for 5 min in PBS
- Wash for 5 min in MilliQ H20
- Dry and mount with Faramount Aqueous Mounting Medium (or any water-soluble mounting media).
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Troubleshooting
Be careful when using PBS together with a AP based detection system as phosphate ions can inhibit the activity of the enzyme.
For DIG System applications do not include MgCl¬2 in the detection buffer as this might lead to spotty background on the slide after the detection procedure.
