Whole mount TUNEL analysis of Xenopus embryos

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  

End labelling

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.