11. Do not blow streams of PBS directly onto the embryo. Clean it
by blowing away from the centre where the embryo is located,
toward the sides of the filter paper.
12. Electroporated embryos can be screened and selected in the
egg, in PBS or in 35 mm petri dishes embedded in the Agar/
Albumen mix. These can be done when preparing the imaging
plates.
13. Dip the bottom of the plates in PBS before transferring them
to the culture chamber to avoid forming bubbles under the
imaging plates.
14. Before sealing the culture chamber and to avoid any condensation during image acquisition, the coverslip should be sprayed
with an antifog solution typically used for scuba diving masks.
15. Place the 6-well plate into the incubation chamber of the
microscope but do not start the video recording straight
away. Leave the embryos to recover for at least 1 h.
16. Define the number of slices, number of tiles, number of channels, laser power and exposure time. Images are acquired in a
mosaic format, with different tiles representing each field
of view.
17. Frequently check the time-lapse image acquisition during the
first hours of imaging as the focus may be lost due to the
embryo settling down and due to its elongation. Refocus as
often as necessary, during the pausing time.
18. In vivo time-lapse imaging of SN cells can be performed using
other systems using some mounting modifications. For example, for confocal imaging we mount the embryos in Lab-Tek
®
2-well glass chamber slides.
19. The Stack focuser plugin detects the sharpest details of each
image in a z-stack.
20. Registration is calculated on the channel displaying the highest
contrast and it is then applied to the other channels.
References
1. Gouignard N, Andrieu C, Theveneau E (2018)
Neural crest delamination and migration: looking forward to the next 150 years. Genesis 56
(6–7):e23107. https://doi.org/10.1002/dvg.
23107
2. Mayor R, Theveneau E (2013) The neural
crest. Development 140(11):2247–2251.
https://doi.org/10.1242/dev.091751
3. Theveneau E, Mayor R (2012) Neural crest
migration: interplay between chemorepellents,
chemoattractants,
contact
inhibition,
epithelial-mesenchymal
transition,
and
collective cell migration. Wiley Interdiscip Rev
Dev Biol 1(3):435–445. https://doi.org/10.
1002/wdev.28
4. Harrington MJ, Hong E, Brewster R (2009)
Comparative analysis of neurulation: first
impressions do not count. Mol Reprod Dev
76(10):954–965. https://doi.org/10.1002/
mrd.21085
5. Lowery LA, Sive H (2004) Strategies of vertebrate neurulation and a re-evaluation of teleost
neural tube formation. Mech Dev 121
In vivo Analysis of the MET During Secondary Neurulation
195
by blowing away from the centre where the embryo is located,
toward the sides of the filter paper.
12. Electroporated embryos can be screened and selected in the
egg, in PBS or in 35 mm petri dishes embedded in the Agar/
Albumen mix. These can be done when preparing the imaging
plates.
13. Dip the bottom of the plates in PBS before transferring them
to the culture chamber to avoid forming bubbles under the
imaging plates.
14. Before sealing the culture chamber and to avoid any condensation during image acquisition, the coverslip should be sprayed
with an antifog solution typically used for scuba diving masks.
15. Place the 6-well plate into the incubation chamber of the
microscope but do not start the video recording straight
away. Leave the embryos to recover for at least 1 h.
16. Define the number of slices, number of tiles, number of channels, laser power and exposure time. Images are acquired in a
mosaic format, with different tiles representing each field
of view.
17. Frequently check the time-lapse image acquisition during the
first hours of imaging as the focus may be lost due to the
embryo settling down and due to its elongation. Refocus as
often as necessary, during the pausing time.
18. In vivo time-lapse imaging of SN cells can be performed using
other systems using some mounting modifications. For example, for confocal imaging we mount the embryos in Lab-Tek
®
2-well glass chamber slides.
19. The Stack focuser plugin detects the sharpest details of each
image in a z-stack.
20. Registration is calculated on the channel displaying the highest
contrast and it is then applied to the other channels.
References
1. Gouignard N, Andrieu C, Theveneau E (2018)
Neural crest delamination and migration: looking forward to the next 150 years. Genesis 56
(6–7):e23107. https://doi.org/10.1002/dvg.
23107
2. Mayor R, Theveneau E (2013) The neural
crest. Development 140(11):2247–2251.
https://doi.org/10.1242/dev.091751
3. Theveneau E, Mayor R (2012) Neural crest
migration: interplay between chemorepellents,
chemoattractants,
contact
inhibition,
epithelial-mesenchymal
transition,
and
collective cell migration. Wiley Interdiscip Rev
Dev Biol 1(3):435–445. https://doi.org/10.
1002/wdev.28
4. Harrington MJ, Hong E, Brewster R (2009)
Comparative analysis of neurulation: first
impressions do not count. Mol Reprod Dev
76(10):954–965. https://doi.org/10.1002/
mrd.21085
5. Lowery LA, Sive H (2004) Strategies of vertebrate neurulation and a re-evaluation of teleost
neural tube formation. Mech Dev 121
In vivo Analysis of the MET During Secondary Neurulation
195