Not Registered?

Products for this protocol

• Alkaline phosphatase buffer (NTMT), made fresh from stocks
   
  
  • 100mM NaCl
     
    
  • 100mM Tris-Cl (pH 9.5)
     
    
  • 50 mM MgCl₂
     
    
  • 1% Tween 20
     
    
• Blocking reagent (Roche, 1096176)
   
  Make a 10% stock of blocking reagent in MAB (no Tween 20) by heating it to dissolve; then autoclave, aliquot, and freeze the stock solution. Blocking reagent works better than "embryo powder" and is easier and more reproducible to make. We have used MABT thereafter for consistency, but maleate does not buffer well, and alternative buffers probably substitute as well or better.
   
  
• Permeabilized Embryos
   
  
• Glutaraldehyde (25%), stored at room temperature
   
  
• 20X SSC
   
  For a 20X solution: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H2O. Adjust the pH to 7.0 with a few drops of a 14-N solution of HCl. Adjust the volume to 1 liter with H2O. Dispense into aliquots. Sterilize by autoclaving. The final concentrations of the ingredients are 3.0 M NaCl and 0.3 M sodium citrate.
   
  
• Hybridization mix
   
  
  • 1.3X SSC
     
    
  • 5 mM EDTA (pH 8.0)
     
    
  • 50% (v/v) Formamide
     
    
  • 0.5% (w/v) CHAPS
     
    
  • 100 µg/mL Heparin
     
    
  • 0.2% Tween 20
     
    
  • 50 µg/mL Yeast RNA
     
    The hybridization mix can be stored at -20°C.
     
    
• MABT (pH 7.5)
   
  
  • 100 mM Maleic acid (pH 7.5)
     
    
  • 150 mM NaCl
     
    
  • 0.1% (v/v) Tween 20
     
    
• Methanol (25%, 50%, and 75%) in PBS-T
   
  
• Paraformaldehyde
   
  
• Proteinase K
   
  
• PBS-T (PBS with 0.1% Tween 20)
   
  
• BM purple AP substrate (Roche)
   
  
• Riboprobes
   
  
• Sheep anti-DIG Fab conjugated to calf intestinal alkaline phosphatase
   
  
• Sheep serum
   
  This serum is used for diluting the antiserum and blocking nonspecific sites in the embryo. Because the anti-DIG is from sheep, it is advisable to use serum from the same species for this step. However, other sera may also give good results. Heat the serum to 70°C for 30 min before use to destroy endogenous alkaline phosphatase activity. If the serum gels (denatures) during heating, it can still be used but must be mixed well after diluting. Alternatively, heat the denatured serum to 55°C only (good results have been obtained by those who have tried it).
   
  
• Sodium azide
   
  

• Microcentrifuge tubes (1.5 mL) or glass vials (4 mL) with screw caps
   
  
• Incubator (rolling or rocking), at 65°C-70°C
   
  
• Suitable containers for handling the embryos:
   
  
  • Reacti-Vials (10 mL), glass (Pierce)
     
    
  • Scintillation vials (5 mL), glass (Fisher)
     
    
  • Screw-capped 2-mL plastic tubes with conical bottoms
     
    
  • Embryos can be collected in a small volume by sucking off the medium.
     
    
• Water bath, at 65°C-70°C
   
  

Day 1: Pretreatments and Hybridization
 

1. Rehydrate embryos through 75%, 50%, and 25% methanol in PBS-T (allowing embryos to settle), and wash twice with PBS-T.
    
   
2. Treat embryos with 10 µg/mL proteinase K in PBS-T as follows:
    
   
   • Treat 6.5-7.5 d post-coitum (dpc) mouse embryos for 5 min.
      
     
   • Treat 8.5 dpc embryos for 10 min.
      
     
   • Treat 9.5 dpc embryos for 15 min.
      
     
   • Treat 10.5 dpc embryos for 20 min.
      
     
   • Decrease proteinase K to 0.5 µg/mL for only 5 min for probes for the apical ectodermal ridge (AER) or superficial tissues such as ectoderm.
      
     The thickness of the tissue determines the strength and length of proteinase K treatment. The thinner the tissue, the more care needs to be taken, and proteinase K treatment may not be required. For thick tissues or organs, a longer proteinase K treatment may be necessary.
      
     
3. Remove proteinase K, rinse the embryos briefly (take care!) with PBS-T, and post-fix for 20 min in 4% paraformaldehyde/0.1% glutaraldehyde in PBS-T.
    
   To guarantee the arrest of proteinase K activity, include 2 mg/mL glycine in the PBS-T washes.
    
   
4. Rinse the embryos, and wash them once with PBS-T. Transfer the embryos to 1.5-mL microcentrifuge tubes or to 4-mL glass vials.
    
   
5. Rinse the embryos once with 1:1 PBS-T/hybridization mix. Let them settle.
    
   
6. Rinse the embryos with 1 mL of hybridization mix. Let them settle.
    
   
7. Replace with 1 mL of fresh hybridization mix, and incubate for 1-24 h at 65°C-70°C.
    
   The embryos can be stored at -20°C before or after prehybridization.
    
   
8. Replace with 1 mL of prewarmed hybridization mix and ~1 µg/mL DIG-labeled RNA probe (as little as 0.1 µg/mL can work). Immediately place at 65°C-70°C.
    
   
9. Incubate for 12-48 h (48 h is preferred) at 65°C-70°C. Gentle rocking or shaking can be useful.
    
   
Day 2: Post-hybridization Washes
 
After each 65°C-70°C wash, let the embryos settle by incubating the tube vertically at 65°C-70°C in a heating block, then change supernatants individually so samples do not cool. Keep wash solutions at 65°C-70°C in a water bath.
 

10. Rinse the embryos twice with prewarmed (65°C-70°C) hybridization mix.
     
    
11. Wash the embryos twice for 30 min each at 65°C-70°C with 1.5 mL of prewarmed hybridization mix.
     
    
12. Wash the embryos for 10 min at 65°C-70°C with 1.5 mL of prewarmed 1:1 hybridization mix/MABT.
     
    
13. Rinse the embryos twice with 1.5 mL of MABT.
     
    
14. Wash the embryos once for 15 min with 1.5 mL of MABT.
     
    
15. Incubate the embryos for 1 h with 1.5 mL of MABT/2% blocking reagent.
     
    
16. Incubate for at least 1 h in 1.5 mL of MABT/2% blocking reagent/20% heat-treated sheep serum.
     
    
17. Incubate overnight at 4°C (or 4 h at room temperature) in 1 mL of fresh MABT/2% blocking reagent/20% sheep serum containing 1/2,000 to 1/10,000 dilution of AP-anti-DIG antibody.
     
    
Day 3: Post-antibody Washes
 

18. Rinse the embryos three times with 1.5 mL of MABT. Transfer them to a glass 4-mL or 20-mL scintillation vial.
     
    
19. Wash the embryos at least five times for 2 to 4 h each and then at least overnight with 10-20 mL of MABT, on a rolling, rotating, or rocking incubator.
     
    Ideally, to lower the background, wash up to 4 d at 4°C in a cold room, with changes of MABT two to three times per day.
     
    
20. Wash the embryos three times for 10-60 min each with 10-20 mL of NTMT.
     
    
Day 4: Histochemistry
 

21. Incubate with enough purple AP substrate to cover embryos at 4°C-10°C for 1-4 d--until the background starts to come up. Wrap vials in foil or keep in a dark drawer during this step.
     
    
22. When color has developed to the desired extent, rinse once and wash at least twice with PBS-T. Refix in 4% paraformaldehyde/0.1% glutaraldehyde/PBS-T for 2 h (at room temperature) or overnight (at 4°C).
     
    
23. Rinse once and wash two times for 10 min with PBS-T. Store at 4°C in PBS-T containing 0.1% azide.