Gautier:TUNEL

Gautier Lab, Columbia University

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Overview

This protocol detects apoptosis patterns in developing Xenopus embryos using TUNEL

Materials

To be added

Procedure

1. Fix embryos (with their vitelline membranes removed) in MEMFA (100mM MOPS, pH 7.4; 2mM EGTA; 1mM MgSO4; 3.7% HCHO) for 60 min
2. Wash 2X for 30 min with MeOH
   • Embryos can then be stored thus in -20 degC
   • I, personally, would not store embryos for TUNEL labelling for longer than one week

Day1 - Terminal Transferase Labeling (performed at room temp)

1. Rehydrate the embryos with 50% MeOH followed by 100% MeOH
2. Wash 2X for 5 min in 1X PBS
3. Wash 1X for 15 min in 1X PBS/0.2% Tween-20
4. Wash 1X for 30 min in 1X PBS/0.5% Tween-20
5. Wash 2X for 10 min in 1X PBS
6. Pre-incubate the embryos in 1X TdT buffer for 60 min
   • TdT buffer supplied by manufacturers is diluted with 1XPBS (NOT with ddH20)
   • 150-200 ul volume should be enough for the 1.9 ml glass vials from FISHER
7. TdT reaction:
   • 1X TdT buffer made up with 1X PBS/0.5uM Dig-dUTP (Roche #1093088) which is 2 ul per ml of reaction vol
   • 150 U/ml reaction vol for rTdT (Invitrogen #10533-065) which is 10 ul per ml of reaction vol
   • incubate o/n at room temp

Day-2 - Addition of anti-Dig Abs

1. Wash embryos 2X for 30 min in 1X PBS/1mM EDTA, 65C
   • I do it in the hybridisation oven
2. Wash 4X for 15 min in 1X PBS, room temp
3. Wash in 1X PBS/0.1% Triton-X with 0.2% BSA, 15 min, r.t.
4. Block with 1X PBS/0.1% Triton-X/0.2% BSA with 20% goat serum, 60 min, r.t.
5. Add anti-Dig at 1:2000 in 1X PBS/0.1% Triton-X/0.2% BSA/20% goat serum
   • I use Fab coupled with alkaline phosphatase (Boehringer Mannheim)
6. Incubate at 4 degC , o/n

Day-3 - Chromogenic Reaction
We find that using alkaline phosphatase with NBT/BCIP as substrates works best for TUNEL; the other chromogenic substrates are a little weak

1. Wash embryos 4X for 30 min in 1X PBS/0.1% Triton-X/0.2% BSA, room temp
2. Wash 2X for 5 min with alkaline phosphatase buffer
   • 100 mM Tris pH9.5, 50 mM MgCl2, 100mM NaCl, 0.1% Tween-20
3. Replace with alkaline phosphatase buffer containing NBT/BCIP for chromogenic reaction
4. Dilute NBT to 75 mg/ml with N-N-dimethylformamide before use
5. BCIP at manufacturer's concentration: 50 mg/ml
6. Add 4.5 ul NBT and 3.5 ul BCIP per ml of buffer for chromogenic reaction
   • I usually use 500 ul reaction vol for each vial
7. Staining (dark purple and punctate) should be apparent 15-30 min; I usually stain for 45-60 min
8. When desired, stop chromogenic reaction with quick rinse in 1X PBS/100mM EDTA
   • very short rinse because the residual BCIP tends to oxidise to a dark coloration which is not good

This is optional, but I usually wash out the background staining i.e. the purple hue in non-staining areas with EtOH.) Dehybrate the embryos in EtOH.

1. Wash in 100% EtOH until satisfied with the staining.
   • Limit EtOH wash to a day max.
2. When satisfied with staining, change to 100% MeOH and wash for 15 min.
3. Rehydrate embryos gradually.
4. Re-fix embryos in MEMFA for at least 4 hours
5. TUNEL staining is best visualised in cleared embryos. The nuclei-staining becomes more apparent in transparent embryos.
6. Dehydrate the embryos in MeOH
   • clear the embryos in BBBA (Benzyl Benzoate: Benzyl Alcohol = 2:1)

Notes

To be added

References

Relevant papers

1. Hensey C and Gautier J. Programmed cell death during Xenopus development: a spatio-temporal analysis. Dev Biol. 1998 Nov 1;203(1):36-48. DOI:10.1006/dbio.1998.9028 |
2. Hensey C and Gautier J. A developmental timer that regulates apoptosis at the onset of gastrulation. Mech Dev. 1997 Dec;69(1-2):183-95. DOI:10.1016/s0925-4773(97)00191-3 |

For sample images: http://www.xenbase.org/other/static/methods/PCD/tunnel-rtn.jsp

Contact

Jean Gautier