imported>Virgilio: moved Veenstra Lab - apoptosis via TUNEL to apoptosis via TUNEL (Veenstra lab)

2011-11-15T19:07:03Z

moved Veenstra Lab - apoptosis via TUNEL to apoptosis via TUNEL (Veenstra lab)

← Older revision	Revision as of 19:07, 15 November 2011
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imported>Virgilio: moved Apoptosis via TUNEL to Veenstra Lab - apoptosis via TUNEL: added lab attribution

2011-11-15T15:52:18Z

moved Apoptosis via TUNEL to Veenstra Lab - apoptosis via TUNEL: added lab attribution

← Older revision	Revision as of 15:52, 15 November 2011
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imported>Pvize: Created page with '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 p…'

2010-01-07T20:49:45Z

Created page with '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 p…'

New page

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. [http://www.xenbase.org/literature/article.do?method=display&articleId=25226]

Or for a fluorescent protocol see Vize et al., 2009. [http://www.xenbase.org/literature/article.do?method=display&articleId=39821]

Apoptosis via TUNEL (Veenstra lab) - Revision history

imported>Virgilio: moved Veenstra Lab - apoptosis via TUNEL to apoptosis via TUNEL (Veenstra lab)

imported>Virgilio: moved Apoptosis via TUNEL to Veenstra Lab - apoptosis via TUNEL: added lab attribution

imported>Pvize: Created page with '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 p…'