in 25
C is preferred, unless the experiment requires a different
temperature.
3. After at least 2 h of collection, change the egg collection cup to
a new apple juice plate. Pour Halocarbon oil 27 on the plate
with collected embryos to identify embryos with the desired
stages.
3.2 Embryo Staging
and Dechorionation
1. Submerged in Halocarbon oil, the interior of the embryos
become visible for recognizing developmental stages. Embryo
preparation alone routinely takes 20 min. Therefore, picking
embryos of sufficiently early stages is necessary. To image
mesoderm-EMT before apical constriction, identify the
embryos just starting cellularization or earlier (Fig. 2). Go
through the plate, pick up around 10–15 embryos of the
desired stages with forceps. If there are too many embryos to
handle while searching around the plate, collect them to a
marked place on the agar plate.
2. Sort through the embryos collected to find the 7–10 best ones
and pick them up together with forceps. Slowly open the
forceps so the embryos are clustered on one leg of the forceps.
Dry the oil on a small piece of paper towel. Leave the cluster of
embryos on the edge of the paper towel in order to clean the oil
on the forceps. Dry the oil as much as possible as it prevents the
bleach from reaching the chorion.
3. Put several drops of 4% sodium hypochlorite/bleach on a small
piece of paper towel. Just enough to cover the texture of the
paper towel. This allows the embryos to completely submerge
in the liquid without running off the paper due to liquid
overflowing.
4. Place the cluster of embryos in the bleach on the paper towel.
Embryos usually get loosened and fall into the bleach once the
cluster touches the bleach.
5. Use the paint brush with a few bristles to separate embryos by
gently sweeping them around and against the paper towel. This
also removes the residual oil on the embryos. From this point
one, the embryos will be kept on this small paper towel until
they are mounted on the glass (see Note 1).
6. While the embryos are being dechorionated, prepare the water
drops to wash embryos. Evenly distribute three large drops of
water around the inside rim of a 60 mm petri dish lid. Make
three such lids with a total of 9 drops of water. Do not leave the
embryos in bleach for more than 1 min.
7. Get embryos out of the bleach by gently picking up the small
piece of paper towel from a corner with forceps. Dry this small
piece of paper towel on regular paper towels. Do not over dry.
Wash the small paper by touching a drop of water, previously
72
Lingkun Gu and Mo Weng
C is preferred, unless the experiment requires a different
temperature.
3. After at least 2 h of collection, change the egg collection cup to
a new apple juice plate. Pour Halocarbon oil 27 on the plate
with collected embryos to identify embryos with the desired
stages.
3.2 Embryo Staging
and Dechorionation
1. Submerged in Halocarbon oil, the interior of the embryos
become visible for recognizing developmental stages. Embryo
preparation alone routinely takes 20 min. Therefore, picking
embryos of sufficiently early stages is necessary. To image
mesoderm-EMT before apical constriction, identify the
embryos just starting cellularization or earlier (Fig. 2). Go
through the plate, pick up around 10–15 embryos of the
desired stages with forceps. If there are too many embryos to
handle while searching around the plate, collect them to a
marked place on the agar plate.
2. Sort through the embryos collected to find the 7–10 best ones
and pick them up together with forceps. Slowly open the
forceps so the embryos are clustered on one leg of the forceps.
Dry the oil on a small piece of paper towel. Leave the cluster of
embryos on the edge of the paper towel in order to clean the oil
on the forceps. Dry the oil as much as possible as it prevents the
bleach from reaching the chorion.
3. Put several drops of 4% sodium hypochlorite/bleach on a small
piece of paper towel. Just enough to cover the texture of the
paper towel. This allows the embryos to completely submerge
in the liquid without running off the paper due to liquid
overflowing.
4. Place the cluster of embryos in the bleach on the paper towel.
Embryos usually get loosened and fall into the bleach once the
cluster touches the bleach.
5. Use the paint brush with a few bristles to separate embryos by
gently sweeping them around and against the paper towel. This
also removes the residual oil on the embryos. From this point
one, the embryos will be kept on this small paper towel until
they are mounted on the glass (see Note 1).
6. While the embryos are being dechorionated, prepare the water
drops to wash embryos. Evenly distribute three large drops of
water around the inside rim of a 60 mm petri dish lid. Make
three such lids with a total of 9 drops of water. Do not leave the
embryos in bleach for more than 1 min.
7. Get embryos out of the bleach by gently picking up the small
piece of paper towel from a corner with forceps. Dry this small
piece of paper towel on regular paper towels. Do not over dry.
Wash the small paper by touching a drop of water, previously
72
Lingkun Gu and Mo Weng