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Murine Whole Mount In Situ Hybridization

Day One

1. Trim embryos and transfer glass scintillation vials.
2. Rehydrate through descending methanol series (75, 50, 25%MEOH in PBT; 10 minutes each )
3. Wash 2X PBT; 10 minutes each.
4. Bleach in 6% hydrogen peroxide in PBT (10 minutes).
5. Wash 3X PBT; 10 minutes each.
6. Permeabilize with Proteinase K Solution (30 ug/ml)in 0.1% PBT at 37°C. Incubate 9.5 minutes for E 10.5 embryos. For larger embryos, treat with proteinase K longer; and for smaller embryos, use a shorter incubation time.
7. To stop digestion, wash 1X with 2 mg/ml glycine in PBT, 10 minutes.
8. Wash 2X PBT, 10 minutes each.
9. Post-fix in 4% paraformaldehyde + 0.2% glutaraldehyde in PBT for 20 minutes at RT.
10. Wash 4X PBT, 10 minutes each.
11. Incubate in pre-hybridization buffer for 2 hours at 70°C.
12. Replace buffer with digoxigenin-labeled riboprobe in pre-hyb.
    buffer at 1ug/ml (only need enough volume to just cover samples ). Incubate overnight at 65°C.

Day Two

1. Wash 3X 30 minutes each at 70°C with Solution 1.
2. Wash 3X 30 minutes each at 70°C with Solution 2.
3. Wash 3X at room temperature, 10 minutes each with TBS-T. (TBS+1% Tween-20)+2mM freshly added levamisole ( 25mg/50ml TBS-T ).
4. Prevent non-specific binding by soaking in Tissue Blocking
   Solution 2-3 hours at room temperature.
5. Incubate overnight at 4°C with anti-DIG antibody (Roche #1093247). Dilute antibody - 1:1500 in Tissue Blocking Solution.

Day Three

1. Wash 3 X at room temperature in TBS-T.
2. Wash 6 X 1 hour each at room temperature in TBS-T.
3. Wash overnight with TBS-T at 4°C, with agitation.

Day Four

1. Wash a few times in TBS-T.

Day Five

1. Wash a few times in TBS-T.

Day Six

1. Wash with TBST 2-3 times
2. Change buffer by washing 3X ,10 minutes each with freshly made NTMT solution.
3. Incubate with Antibody Detection Solution. Keep the reaction covered in dark.
4. Developing reaction may take a few hours to a few days.

After Color Develop

1. Wash 3 - 4 X PBT for 10-30 min total.
2. Fix in 4% PF/ 0.2% glutaraldehyde in PBS for 2 hours at RT or O/N at 4°C.
3. Rinse 1X and Wash 10min X3 with PBT.
4. Dehydrate 25, 50, 70, 100% methanol in PBT 5min each.
5. Rehydrate 70, 50, 25% methanol in PBT 5min each.
6. PBT 3min X2
7. Clear: 1:1 Glycerol : PBT for 1hour.
8. 4:1 Glycerol : PBT for 1hour.
9. Store at 4°C.

 

SOLUTIONS

To avoid RNAse contamination, all solutions should be made in baked glassware or in sterile plastic tubes ( e.g , 15 or 50 ml ).

Use Rnase free ddH₂O.

Bleaching Solutions (make fresh every time)
6% H₂O2 in PBS-T.

Proteinase K solution (Roche #745723 )
Prepare a 10 mg/ml stock in 0.1 M Tyis-HCl ( pH 7.5 ). Store in small aliquots at -20°C.
Dilute in PBS-T just before use.

Yeast tRNA (Roche 100mg )
Add 4 ml nuclease - free water to the 100mg bottle and mix.
Dispense into 1 ml aliquots;
store at -20°C.

Prehybridization Buffer

Total Volume 50ml 200ml

Deionized formamide ( 50% ) 25ml 100ml
20X SSC ( 5X ) 12,5ml 50ml
10% SDS 5ml 20ml
Yeast tRNA (50 mg/ml ) 100ul 400ul
Heparin (50ug/ml ) 2.5mg 10mg
ddH₂O 7.5ml 30ml
pH 4.5 and freeze aliquots at -20°C.

Solution 1

Total Volume 50ml 200ml

deionized formamide ( 50% ) 25ml 100ml
20 X SSC ( 5X ) 12.5ml 50ml
10% SDS (1% ) 5ml 20ml
ddH₂O 7.5ml 30ml
pH to 4.5 and freeze aliquots at -20°C.

Solution 2

Total volume: 50ml 200ml

Deionized formamide ( 50% ) 25ml 100ml
20X SSC ( 2X ) 5ml 20ml
ddH₂O 20ml 80ml
pH to 4.5 and freeze aliquots at -20°C.

10X TBS-T

Total Volume 1000ml

NaCl 80g
Kcl 2g
1M Tris-Hcl pH 7.5 250ml
Tween-20 110g
ddH₂O 640ml

Tissue Blocking Solution (make fresh every time )

Total Volume 10ml

2% blocking solution ( see below ) 8ml
Head Inactivated Goat Serum ( see below ) 2ml

2% Blocking Solution

Total Volume 50ml

Blocking reagent (Roche # 1096176 ) 1g
1M Tris-HCl pH7.5 5ml
5M NaCl 1.5ml
ddH₂O 43.5ml

Heat Inactivated Goat Serum
Heat normal goat serum to 65 C in a water bath for 15 minutes and freeze aliquots at -20°C.

NTMT (make fresh 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 41ml

Antibody Detection Solution (make fresh every time)

Total volume: 10ml

NTMT 10ml
NBT (Roche#1383213) 50 ul
(final 0.5 mg/ml)
BCIP (Roche#1383221) 38ul
(final 0.1 mg/ml )

 

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