5. Finally, correct the drift along the time-lapse video using the
MultiStackReg plugin that is based on the StackReg plugin (see
Note 20). The best focused time-lapse movie is now aligned
(Fig. 5a, b, Movie S2).
6. Individual cells are followed spatiotemporally using the Manual
Tracking plugin of the Image J software (Fig. 5c, d, Movie S3).
4 Notes
1. It is best to perform steps 4 and 5 before placing the electroporation mixture on the epiblast to avoid its diffusion.
2. Repeated injection of DNA through the membranes may block
the needle, in which case, clean the tip with a wet tissue or
cotton swab.
3. The electroporation mixture can be passed over the top of the
epiblast by blowing air through the aspirator tube or with the
help of a syringe.
4. Maintain the electrodes moist with 1% of P/S in MilliQ H 2 O
to diminish the resistance between the electrodes.
5. To prepare the imaging plates, collect the thin albumen at the
time of electroporation with a syringe, as both procedures are
performed on the same day. Thin albumen can also be collected
by cracking an egg incubated overnight into a petri dish and
recovering it with a Pasteur pipette.
6. Wait for the two solutions to equilibrate to the same temperature as the hot agar could “cook” the thin albumen if they are
mixed too early.
7. Try to fill the culture inserts in a way that the culture medium
dries as flat as possible, avoiding the introduction of bubbles.
8. Imaging plates should be prepared fresh, no more than 2 days
in advance. The plates can be stored at 4
C in sterile 35 mm
petri dishes.
9. The albumen prevents the membranes from attaching to the
filter paper so try to remove as much as possible. However, if
the embryo is already dry and it is touched again with the
tissue, the blastoderm may attach and break and the embryo
will be lost.
10. Pull the filter paper with the embryo away from the yolk
obliquely. It is best to pull in the direction of the yolk flow
produced by the cutting of membranes or along the anteroposterior embryonic axis.
194
Elena Gonzalez-Gobartt et al.
MultiStackReg plugin that is based on the StackReg plugin (see
Note 20). The best focused time-lapse movie is now aligned
(Fig. 5a, b, Movie S2).
6. Individual cells are followed spatiotemporally using the Manual
Tracking plugin of the Image J software (Fig. 5c, d, Movie S3).
4 Notes
1. It is best to perform steps 4 and 5 before placing the electroporation mixture on the epiblast to avoid its diffusion.
2. Repeated injection of DNA through the membranes may block
the needle, in which case, clean the tip with a wet tissue or
cotton swab.
3. The electroporation mixture can be passed over the top of the
epiblast by blowing air through the aspirator tube or with the
help of a syringe.
4. Maintain the electrodes moist with 1% of P/S in MilliQ H 2 O
to diminish the resistance between the electrodes.
5. To prepare the imaging plates, collect the thin albumen at the
time of electroporation with a syringe, as both procedures are
performed on the same day. Thin albumen can also be collected
by cracking an egg incubated overnight into a petri dish and
recovering it with a Pasteur pipette.
6. Wait for the two solutions to equilibrate to the same temperature as the hot agar could “cook” the thin albumen if they are
mixed too early.
7. Try to fill the culture inserts in a way that the culture medium
dries as flat as possible, avoiding the introduction of bubbles.
8. Imaging plates should be prepared fresh, no more than 2 days
in advance. The plates can be stored at 4
C in sterile 35 mm
petri dishes.
9. The albumen prevents the membranes from attaching to the
filter paper so try to remove as much as possible. However, if
the embryo is already dry and it is touched again with the
tissue, the blastoderm may attach and break and the embryo
will be lost.
10. Pull the filter paper with the embryo away from the yolk
obliquely. It is best to pull in the direction of the yolk flow
produced by the cutting of membranes or along the anteroposterior embryonic axis.
194
Elena Gonzalez-Gobartt et al.