This protocol detects apoptosis patterns in developing Xenopus embryos using TUNEL
To be added
- Fix embryos (with their vitelline membranes removed) in MEMFA (100mM MOPS, pH 7.4; 2mM EGTA; 1mM MgSO4; 3.7% HCHO) for 60 min
- 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)
- Rehydrate the embryos with 50% MeOH followed by 100% MeOH
- Wash 2X for 5 min in 1X PBS
- Wash 1X for 15 min in 1X PBS/0.2% Tween-20
- Wash 1X for 30 min in 1X PBS/0.5% Tween-20
- Wash 2X for 10 min in 1X PBS
- 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
- 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
- Wash embryos 2X for 30 min in 1X PBS/1mM EDTA, 65C
- I do it in the hybridisation oven
- Wash 4X for 15 min in 1X PBS, room temp
- Wash in 1X PBS/0.1% Triton-X with 0.2% BSA, 15 min, r.t.
- Block with 1X PBS/0.1% Triton-X/0.2% BSA with 20% goat serum, 60 min, r.t.
- 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)
- 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
- Wash embryos 4X for 30 min in 1X PBS/0.1% Triton-X/0.2% BSA, room temp
- Wash 2X for 5 min with alkaline phosphatase buffer
- 100 mM Tris pH9.5, 50 mM MgCl2, 100mM NaCl, 0.1% Tween-20
- Replace with alkaline phosphatase buffer containing NBT/BCIP for chromogenic reaction
- Dilute NBT to 75 mg/ml with N-N-dimethylformamide before use
- BCIP at manufacturer's concentration: 50 mg/ml
- 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
- Staining (dark purple and punctate) should be apparent 15-30 min; I usually stain for 45-60 min
- 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.
- Wash in 100% EtOH until satisfied with the staining.
- Limit EtOH wash to a day max.
- When satisfied with staining, change to 100% MeOH and wash for 15 min.
- Rehydrate embryos gradually.
- Re-fix embryos in MEMFA for at least 4 hours
- TUNEL staining is best visualised in cleared embryos. The nuclei-staining becomes more apparent in transparent embryos.
- Dehydrate the embryos in MeOH
- clear the embryos in BBBA (Benzyl Benzoate: Benzyl Alcohol = 2:1)
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For sample images: http://www.xenbase.org/other/static/methods/PCD/tunnel-rtn.jsp