Tunel staining for Whole Embryos

TUNEL Staining for whole mount embryos of

TUNEL Staining for Whole Embryos:

*IMPORTANT: Use PBST – tween 0.1% for steps 1-8

Use PBST – Triton-X for steps 9-14

1. 
   Fix embryos with 4% paraformaldehyde in PBS for 24 hrs. Best if using fresh paraformaldehyde.
   
2. 
   Wash embryos twice in PBS, 5 min each.
   
3. 
   Transfer embryos into a Petri dish and dechorionate using glass forceps.
   
4. 
   Transfer embryos into 1.5ml eppendorf tubes (microcentrifuge tubes) containing 50% methanol for 5 min.
   
5. 
   Replace 50% methanol with 100% methanol for 5 minutes . After 5 minutes, replace with fresh 100% MetOH.
   
6. 
   a) Store microcentrifuge tubes in -20 for 12 hrs or until ready for use.
   b) Acetone wash for 20 minutes.
   
7. 
   a) Re-hydrate embryos with 1 mL as follows for 5 min each:
   
   1. 
      75% methanol (37.5 mL) + 25% PBST (12.5 mL)
      
   2. 
      50% methanol (25 mL) + 50% PBST (25 mL)
      
   3. 
      25% methanol (12.5 mL) + 75% PBST (37.5 mL)
      
8. 
   a) Wash embryos with 100% PBST (Tween) 2 X 5 min.
   b) Proteinase K wash for 5 minutes.
   c) Fix PFA for 20 minutes. Go back to step 8.
   
9. 
   Wash embryos for at least 1 hr (3 X 20 mins) with PBST (PBS, 1% Triton-X).
   
   1. 
      During the third wash, prepare TUNEL reaction
      
      1. 
         Remove 50 uL Label Solution (vial 2) to serve as negative control
         
      2. 
         Add 25 ul of Enzyme solution (vial 1) to 225 ul Labeled Solution in a new microcentrifuge tube (total 250 ul volume); make sure to mix well by pippetting up and down several times
         
      3. 
         Leave on ice until ready for use
         
10. 
    Wash embryos 2 X quickly with PBS.
    
11. 
    Put embryos in foil covered 1.5 mL eppendor tube and add 50 uL TUNEL rxn mixture per tube. For negative control add 50 uL label solution.
    
12. 
    Incubate for 60 min @ 37C
    
13. 
    Wash samples 2 X with PBS for 5 min each time and store embryos in PBS.
    
14. 
    Analyze samples with flourecescent microscope using 0.8% low melting agarose
    

Notes: 20 ng/mL  dilute 1000X to get 20 ug/mL  1 uL (of 20 ng/mL) + 999 uL H₂O

Revision 01/02/07