8. Optional: Depending on the experiment, embryos can be
injected then electroporated with DNA vectors in combination
with or instead of DiI. For electroporation protocol, see [6].
9. Incubate embryos until desired developmental stage.
3.3 Create
a Few Tools
To assist with mounting embryos on EC culture, create paper
washers following steps 1 and 2:
1. Trace coin on Whatman filter paper with pencil and cut circle
with fine scissors.
2. Cut out center hole in paper leaving an approximately 0.5 cm
ring of paper. Keep paper clean and dry.
A large environmental chamber around the entire stage,
objective and condenser unit is useful to eliminate thermal drift
of the sample. However, without a small chamber inside to
keep the sample hydrated, the EC culture medium will dehydrate and constantly change the focal plane of the embryo or its
health will fail. To construct this small chamber, follow steps 3
and 4.
3. Cut sheets of plastic to make a box with a bottom the same size
as
the
microscope
stage
insert
approximately
13 Â 11 Â 2.5 cm. The lid of the box should be removable
with a single hole roughly the same size as the objective to be
used. Lids can be created for various objectives but we do not
recommend using an immersion objective for this protocol (see
Note 2).
4. Seal the lid to the bottom with vacuum grease carefully expelled
from a 10 mL syringe. In addition, humidity can be added to
the chamber by incorporating a slow flow of humidified air
through a bubbler or wet paper towels within the chamber.
3.4 Mounting
Embryos and Imaging
1. Turn on the microscope and environmental chamber to evenly
heat components to 37
C at least 2 h before the samples are
ready. Pre-warm EC culture dishes to 37
C, 30 min before
working with embryos.
2. Screen embryos for health, stage, and proper labeling. Neural
crest EMT events happen over several hours in the trunk, but to
stage match experiments we recommend choosing an embryo
where the neural tube is well labeled, anterior neural crest can
be observed to be exiting the neural tube but focus the imaging
on a somite level where the neural crest has yet to emerge. This
way, all embryos from different days of experiments can be
time-aligned to the first neural crest cell to exit.
3. Open egg and create a large window for manipulation. Place
one paper ring centered over embryo (Fig. 1a). While the ring
is resting on the embryo, peel back more vitelline membrane
110
Mary Cathleen McKinney and Paul M. Kulesa
injected then electroporated with DNA vectors in combination
with or instead of DiI. For electroporation protocol, see [6].
9. Incubate embryos until desired developmental stage.
3.3 Create
a Few Tools
To assist with mounting embryos on EC culture, create paper
washers following steps 1 and 2:
1. Trace coin on Whatman filter paper with pencil and cut circle
with fine scissors.
2. Cut out center hole in paper leaving an approximately 0.5 cm
ring of paper. Keep paper clean and dry.
A large environmental chamber around the entire stage,
objective and condenser unit is useful to eliminate thermal drift
of the sample. However, without a small chamber inside to
keep the sample hydrated, the EC culture medium will dehydrate and constantly change the focal plane of the embryo or its
health will fail. To construct this small chamber, follow steps 3
and 4.
3. Cut sheets of plastic to make a box with a bottom the same size
as
the
microscope
stage
insert
approximately
13 Â 11 Â 2.5 cm. The lid of the box should be removable
with a single hole roughly the same size as the objective to be
used. Lids can be created for various objectives but we do not
recommend using an immersion objective for this protocol (see
Note 2).
4. Seal the lid to the bottom with vacuum grease carefully expelled
from a 10 mL syringe. In addition, humidity can be added to
the chamber by incorporating a slow flow of humidified air
through a bubbler or wet paper towels within the chamber.
3.4 Mounting
Embryos and Imaging
1. Turn on the microscope and environmental chamber to evenly
heat components to 37
C at least 2 h before the samples are
ready. Pre-warm EC culture dishes to 37
C, 30 min before
working with embryos.
2. Screen embryos for health, stage, and proper labeling. Neural
crest EMT events happen over several hours in the trunk, but to
stage match experiments we recommend choosing an embryo
where the neural tube is well labeled, anterior neural crest can
be observed to be exiting the neural tube but focus the imaging
on a somite level where the neural crest has yet to emerge. This
way, all embryos from different days of experiments can be
time-aligned to the first neural crest cell to exit.
3. Open egg and create a large window for manipulation. Place
one paper ring centered over embryo (Fig. 1a). While the ring
is resting on the embryo, peel back more vitelline membrane
110
Mary Cathleen McKinney and Paul M. Kulesa