4. Image5D
(Joachim
Walter—https://imagej.nih.gov/ij/
plugins/image5d.html) plugins.
5. Stack focuser (Michael Umorin–https://imagej.nih.gov/ij/
plugins/stack-focuser.html) plugin.
6. MultiStackReg plugin (Brad Busse and Kota Miura—http://
bradbusse.net/downloads.html).
7. StackReg plugin [42] (http://bigwww.epfl.ch/thevenaz/
stackreg/).
8. Grid/Collection stitching plugin [43] (https://imagej.net/
Image_Stitching#Grid.2FCollection_Stitching).
9. Manual Tracking plugin (Fabrice P. Cordelie `res—https://
imagej.nih.gov/ij/plugins/track/track.html).
3 Methods
3.1 Electroporation
of Chick SN Cells
In Ovo
1. Incubate eggs horizontally at 38.5
C in an atmosphere of 70%
humidity until stage HH9.
2. Remove 5 mL of albumen from the egg with a syringe.
3. Open a window at the top of the shell with curved scissors to
visualize the embryo.
4. Make a small hole with thin forceps in the posterior region of
the area opaca, just outside of the area pellucida. Avoid touching the embryo (Fig. 2a).
5. Pour 200 mL of 1% P/S onto the embryo to improve electrode
conductivity (see Note 1).
6. Inject the DNA solution onto the epiblast with a glass capillary
by blowing air through the aspirator tube. Introduce the DNA
into the small concave region at their posterior end of the stage
HH9 embryo, where the neural tube is still open (Fig. 2a, b: see
Notes 2 and 3).
7. Carefully insert the platinum electrode connected to the positive lead (+) below the embryo through the hole made previously, parallel to its anteroposterior axis (Fig. 2a, b).
8. Position the tungsten microelectrode connected to the negative lead (À) on top of the embryo, also parallel to the embryo’s
anteroposterior axis (Fig. 2a, b).
9. Deliver five 50 ms square pulses of 5 V at intervals of 50 ms with
the electroporator (see Note 4).
10. Seal the window in the shell with tape and incubate embryos
until they reach stage HH15 (+24 h).
In vivo Analysis of the MET During Secondary Neurulation
191
(Joachim
Walter—https://imagej.nih.gov/ij/
plugins/image5d.html) plugins.
5. Stack focuser (Michael Umorin–https://imagej.nih.gov/ij/
plugins/stack-focuser.html) plugin.
6. MultiStackReg plugin (Brad Busse and Kota Miura—http://
bradbusse.net/downloads.html).
7. StackReg plugin [42] (http://bigwww.epfl.ch/thevenaz/
stackreg/).
8. Grid/Collection stitching plugin [43] (https://imagej.net/
Image_Stitching#Grid.2FCollection_Stitching).
9. Manual Tracking plugin (Fabrice P. Cordelie `res—https://
imagej.nih.gov/ij/plugins/track/track.html).
3 Methods
3.1 Electroporation
of Chick SN Cells
In Ovo
1. Incubate eggs horizontally at 38.5
C in an atmosphere of 70%
humidity until stage HH9.
2. Remove 5 mL of albumen from the egg with a syringe.
3. Open a window at the top of the shell with curved scissors to
visualize the embryo.
4. Make a small hole with thin forceps in the posterior region of
the area opaca, just outside of the area pellucida. Avoid touching the embryo (Fig. 2a).
5. Pour 200 mL of 1% P/S onto the embryo to improve electrode
conductivity (see Note 1).
6. Inject the DNA solution onto the epiblast with a glass capillary
by blowing air through the aspirator tube. Introduce the DNA
into the small concave region at their posterior end of the stage
HH9 embryo, where the neural tube is still open (Fig. 2a, b: see
Notes 2 and 3).
7. Carefully insert the platinum electrode connected to the positive lead (+) below the embryo through the hole made previously, parallel to its anteroposterior axis (Fig. 2a, b).
8. Position the tungsten microelectrode connected to the negative lead (À) on top of the embryo, also parallel to the embryo’s
anteroposterior axis (Fig. 2a, b).
9. Deliver five 50 ms square pulses of 5 V at intervals of 50 ms with
the electroporator (see Note 4).
10. Seal the window in the shell with tape and incubate embryos
until they reach stage HH15 (+24 h).
In vivo Analysis of the MET During Secondary Neurulation
191