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.
