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Gautier Lab, Columbia University

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This protocol detects apoptosis patterns in developing Xenopus embryos using TUNEL


To be added


  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)


To be added


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 | PubMed ID:9806771 | HubMed [Paper1]
  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 | PubMed ID:9486540 | HubMed [Paper2]

All Medline abstracts: PubMed | HubMed

For sample images:


Jean Gautier