Gautier:TUNEL

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
 * 3) * Embryos can then be stored thus in -20 degC
 * 4) * 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
 * 7) *TdT buffer supplied by manufacturers is diluted with 1XPBS (NOT with ddH20)
 * 8) *150-200 ul volume should be enough for the 1.9 ml glass vials from FISHER
 * 9) TdT reaction:
 * 10) *1X TdT buffer made up with 1X PBS/0.5uM Dig-dUTP (Roche #1093088) which is 2 ul per ml of reaction vol
 * 11) *150 U/ml reaction vol for rTdT (Invitrogen #10533-065) which is 10 ul per ml of reaction vol
 * 12) *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
 * 2) *I do it in the hybridisation oven
 * 3) Wash 4X for 15 min in 1X PBS, room temp
 * 4) Wash in 1X PBS/0.1% Triton-X with 0.2% BSA, 15 min, r.t.
 * 5) Block with 1X PBS/0.1% Triton-X/0.2% BSA with 20% goat serum, 60 min, r.t.
 * 6) Add anti-Dig at 1:2000 in 1X PBS/0.1% Triton-X/0.2% BSA/20% goat serum
 * 7) *I use Fab coupled with alkaline phosphatase (Boehringer Mannheim)
 * 8) 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

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 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
 * 3) *100 mM Tris pH9.5, 50 mM MgCl2, 100mM NaCl, 0.1% Tween-20
 * 4) Replace with alkaline phosphatase buffer containing NBT/BCIP for chromogenic reaction
 * 5) Dilute NBT to 75 mg/ml with N-N-dimethylformamide before use
 * 6) BCIP at manufacturer's concentration: 50 mg/ml
 * 7) Add 4.5 ul NBT and 3.5 ul BCIP per ml of buffer for chromogenic reaction
 * 8) *I usually use 500 ul reaction vol for each vial
 * 9) Staining (dark purple and punctate) should be apparent 15-30 min; I usually stain for 45-60 min
 * 10) When desired, stop chromogenic reaction with quick rinse in 1X PBS/100mM EDTA
 * 11) *very short rinse because the residual BCIP tends to oxidise to a dark coloration which is not good
 * 1) Wash in 100% EtOH until satisfied with the staining.
 * 2) *Limit EtOH wash to a day max.
 * 3) When satisfied with staining, change to 100% MeOH and wash for 15 min.
 * 4) Rehydrate embryos gradually.
 * 5) Re-fix embryos in MEMFA for at least 4 hours
 * 6) TUNEL staining is best visualised in cleared embryos. The nuclei-staining becomes more apparent in transparent embryos.
 * 7) Dehydrate the embryos in MeOH
 * 8) *clear the embryos in BBBA (Benzyl Benzoate: Benzyl Alcohol = 2:1)

Contact
Jean Gautier