larger paper rings may be needed. If possible, cut alongside of
paper ring while cutting out embryo to crimp edges of paper to
the tissue. Dull or wide forceps are best for holding the free side
of embryo while you are cutting. Attempt to only hold tissue
outside the vascular plexus.
5. Immediately transfer embryo dorsal side up to a warm
EC culture dish (Fig. 1b). If the embryo has disengaged with
the filter paper, see Note 3 about remounting. It is essential
that the extra-embryonic tissue is stretched tightly in a circle or
the tissue will not develop properly.
6. If too many yolk particles have transferred with the embryo,
gently drop Ringer’s solution just to the side of the embryo and
remove yolk with a narrow-tipped pipette. Avoid directly
touching the embryo. See Note 4 about yolk and Note 5
about excess water in the dish.
7. Replace lid to EC culture dish and place in incubator and
mount a few embryos in case an embryo dies or is labeled
poorly.
8. Optional: Multiple embryos can be placed in one EC culture
dish for multi-positional imaging see Note 6.
9. Place EC culture dish without lid into the small box on the
stage of the microscope (Fig. 1c–e). Seal the lid of the box with
vacuum grease and place parafilm around objective to reduce
the gap between the objective and lid. It will probably not be
airtight since some flexibility is needed to move the objective
for a z-stack but air flow should be reduced enough to slow
dehydration.
10. Set up imaging parameters for your label and region of interest
on the microscope (Fig. 1f–i). Take great care to not allow
contact between the objective and the embryo. If the objective
pulls on the tissue, the embryo will either tear or not lay evenly
taut and thus not develop properly. Clean the objective and
start with a new embryo.
11. The embryo in this environment is fragile and should be
exposed to a minimum of laser light. EMT events occur at
the dorsal most part of the neural tube but you may wish to
image the neural crest as they migrate deeper into the tissue but
the more deeply you image, the longer the image will take to
acquire. Less than 1 min of continuous excitation is ideal.
Frequency of images should be more than double the amount
of time exposed to the laser, i.e., 1 min of imaging, 2 min or
more of rest. For the best automated tracking, images should
be taken frequently enough that a cell at least partially overlaps
its position in the previous image. Longer time intervals are
fine but will require more hand-corrections to the track.
Depending on what type of information you require, you
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Mary Cathleen McKinney and Paul M. Kulesa
paper ring while cutting out embryo to crimp edges of paper to
the tissue. Dull or wide forceps are best for holding the free side
of embryo while you are cutting. Attempt to only hold tissue
outside the vascular plexus.
5. Immediately transfer embryo dorsal side up to a warm
EC culture dish (Fig. 1b). If the embryo has disengaged with
the filter paper, see Note 3 about remounting. It is essential
that the extra-embryonic tissue is stretched tightly in a circle or
the tissue will not develop properly.
6. If too many yolk particles have transferred with the embryo,
gently drop Ringer’s solution just to the side of the embryo and
remove yolk with a narrow-tipped pipette. Avoid directly
touching the embryo. See Note 4 about yolk and Note 5
about excess water in the dish.
7. Replace lid to EC culture dish and place in incubator and
mount a few embryos in case an embryo dies or is labeled
poorly.
8. Optional: Multiple embryos can be placed in one EC culture
dish for multi-positional imaging see Note 6.
9. Place EC culture dish without lid into the small box on the
stage of the microscope (Fig. 1c–e). Seal the lid of the box with
vacuum grease and place parafilm around objective to reduce
the gap between the objective and lid. It will probably not be
airtight since some flexibility is needed to move the objective
for a z-stack but air flow should be reduced enough to slow
dehydration.
10. Set up imaging parameters for your label and region of interest
on the microscope (Fig. 1f–i). Take great care to not allow
contact between the objective and the embryo. If the objective
pulls on the tissue, the embryo will either tear or not lay evenly
taut and thus not develop properly. Clean the objective and
start with a new embryo.
11. The embryo in this environment is fragile and should be
exposed to a minimum of laser light. EMT events occur at
the dorsal most part of the neural tube but you may wish to
image the neural crest as they migrate deeper into the tissue but
the more deeply you image, the longer the image will take to
acquire. Less than 1 min of continuous excitation is ideal.
Frequency of images should be more than double the amount
of time exposed to the laser, i.e., 1 min of imaging, 2 min or
more of rest. For the best automated tracking, images should
be taken frequently enough that a cell at least partially overlaps
its position in the previous image. Longer time intervals are
fine but will require more hand-corrections to the track.
Depending on what type of information you require, you
112
Mary Cathleen McKinney and Paul M. Kulesa