TUNEL Staining for Whole Embryos:
*IMPORTANT: Use PBST – tween 0.1% for steps 1-8
Use PBST – Triton-X for steps 9-14
-
Fix embryos with 4% paraformaldehyde in PBS for 24 hrs. Best if using fresh paraformaldehyde.
-
Wash embryos twice in PBS, 5 min each.
-
Transfer embryos into a Petri dish and dechorionate using glass forceps.
-
Transfer embryos into 1.5ml eppendorf tubes (microcentrifuge tubes) containing 50% methanol for 5 min.
-
Replace 50% methanol with 100% methanol for 5 minutes . After 5 minutes, replace with fresh 100% MetOH.
-
a) Store microcentrifuge tubes in -20 for 12 hrs or until ready for use.
b) Acetone wash for 20 minutes.
-
a) Re-hydrate embryos with 1 mL as follows for 5 min each:
-
75% methanol (37.5 mL) + 25% PBST (12.5 mL)
-
50% methanol (25 mL) + 50% PBST (25 mL)
-
25% methanol (12.5 mL) + 75% PBST (37.5 mL)
-
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.
-
Wash embryos for at least 1 hr (3 X 20 mins) with PBST (PBS, 1% Triton-X).
-
During the third wash, prepare TUNEL reaction
-
Remove 50 uL Label Solution (vial 2) to serve as negative control
-
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
-
Leave on ice until ready for use
-
Wash embryos 2 X quickly with PBS.
-
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.
-
Incubate for 60 min @ 37C
-
Wash samples 2 X with PBS for 5 min each time and store embryos in PBS.
-
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
Dostları ilə paylaş: |