imported>Pvize |
imported>Pvize |
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| See:
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| Veenstra et al. (1998) Cell Death Diff. 5:774-784
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| protocol adapted from Blaschke et al., (1996) Development 122, 1165-1174. Submitted by Gert Veenstra.
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|
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| '''Fixation and pretreatment'''
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|
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| Dejelly albino embryos carefully in 2% Cysteine (pH 7.8).
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| Remove the vitellin membrane with two pairs of tweezers (or carefully pierce multiple times after fixation)
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| Fix the embryos in MEMPFA for 1 hour at room temperature
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| (MEMPFA: 100 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde)
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| Wash 2 times 30 minutes in methanol, store in methanol at -20 °C.
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| Wash embryos 2 times 15 minutes at room temperature in PBT
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| (0.2% Tween in phosphate buffered saline (PBS))
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|
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| Wash embryos 2 times 15 minutes at room temperature in PBS
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|
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|
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| '''End labelling'''
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| Wash embryos 30 min. in TdT buffer (Gibco) 1 hr. at room temp.
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| Incubate embryos overnight at room temp., in TdT buffer containing 0.5 µM digoxygenin-dUTP (Boehringer) and 150 U/ml TdT (Gibco).
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| Wash 2 x 1 hr. with PBS/EDTA (1 mM), at 65°C
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| Wash 4 x 1 hr. with PBS at room temp.
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|
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| '''Detection and chromogenic reaction'''
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| As for in situ hybridization, see:
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|
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| 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. [http://www.xenbase.org/literature/article.do?method=display&articleId=25226]
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|
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| Or for a fluorescent protocol see Vize et al., 2009. [http://www.xenbase.org/literature/article.do?method=display&articleId=39821]
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