Apoptosis via TUNEL (Veenstra lab)

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See: 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.

Fixation and pretreatment

Dejelly albino embryos carefully in 2% Cysteine (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 situ hybridization: An improved whole mount method for Xenopus embryos, vol. 36. Academic Press Inc., San Diego, pp. 685-695. [1]

Or for a fluorescent protocol see Vize et al., 2009. [2]