Veenstra et al. (1998) Cell Death Diff. 5:774-784
protocol adapted from Blaschke et al., (1996) Development 122, 1165-1174. Submitted by Gert Veenstra.
To view images of PCD patterns in embryos stained with TUNEL please visit the pages provided by Veenstra and by Gautier.
Fixation and pretreatment
Dejelly albino embryos carefully in 2% Cystein (pH 7.8).
Remove the vitellin membrane with two pairs of tweezers
(or carefully pierce multiple times after fixation)
Fix the embryos in MEMPFA for 1 hour at room temperature
(MEMPFA: 100 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde)
Wash 2 times 30 minutes in methanol, store in methanol at -20 °C.
Wash embryos 2 times 15 minutes at room temperature in PBT
(0.2% Tween in phosphate buffered saline (PBS))
Wash embryos 2 times 15 minutes at room temperature in PBS
Wash embryos 30 min. in TdT buffer (Gibco) 1 hr. at room temp.
Incubate embryos overnight at room temp., in TdT buffer containing
0.5 µM digoxygenin-dUTP (Boehringer) and
150 U/ml TdT (Gibco).
Wash 2 x 1 hr. with PBS/EDTA (1 mM), at 65°C
Wash 4 x 1 hr. with PBS at room temp.
Detection and chromogenic reaction
As for in situ hybridization, see:
Harland,R.M. (1991) In Kay,B.K. and Peng,H.B. (eds.), In situ hybridization: An improved whole mount method for Xenopus embryos, vol. 36. Academic Press Inc., San Diego, pp. 685-695.