7. Combine water and yeast in small quantities in a tube, while
continuously mixing with a spatula until the consistency resembles that of smooth peanut butter. Store the tube on a tray
(to prevent spills) at 4
C. The lid of the Falcon tube should
only be partially closed, for as the yeast ferments, carbon dioxide is produced, and the tube can burst if the gas cannot be
released.
8. Embryos can be collected throughout the day or overnight,
and selected under the stereomicroscope based on their morphology [33]; or collections can be timed and embryos can be
aged until they reach the appropriate developmental stage.
Development is 1.8 times slower at 18
C than at 25
C [34],
a fact that can be used to facilitate experiments. For example, to
select embryos at stage 14–15 of embryonic development for
wound healing assays, collect embryos for 2 h and age the plate
for 10 h at 25
C or for 18 h at 18
C. dsRNA injections must
be conducted in syncytial embryos, before cells form, and thus,
embryos must be collected for 30–60 min and immediately
prepared for injection.
9. Two strips of plasticine can be used as supports to facilitate
retrieval of the coverslip from the Petri dish (Fig. 1e).
10. To avoid desiccation, do not spend more than 5 min aligning
embryos. At the end, use the back of the forceps to remove all
the non-aligned embryos from the agar block so that they are
not transferred to the coverslip.
11. To image the ventral epidermis in an inverted microscope (e.g.,
for wound healing assays), orient embryos with their convex
(ventral) side up (Fig. 1f, left). To image dorsally (e.g., to
image dorsal closure or cardiac morphogenesis), the convex
side of the embryo should face the agar block (Fig. 1f, right).
12. If embryos burst when the oxygen-permeable membrane is
placed on top of the coverslip, or if the oil does not spread to
cover the entire coverslip, repeat using an additional drop of
oil. On the contrary, if embryos float and move in between the
membrane and the coverslip, reduce the amount of oil.
13. Dehydration times will be longer when humidity is high. Begin
with 5 min. If embryos are plump and the vitelline membrane is
hard to pierce with a microneedle, increase the dehydration
time in 1-min increments. If the vitelline membrane forms
folds or does not slightly resist deformation by the needle,
decrease the dehydration time.
14. For microinjections, it is often preferable to use only halocarbon oil 700 without mixing with halocarbon oil 27. The
greater viscosity of halocarbon oil 700 helps preserve the
integrity of the embryo and prevent “leaks” during the injection procedure.
214
Gordana Scepanovic et al.
continuously mixing with a spatula until the consistency resembles that of smooth peanut butter. Store the tube on a tray
(to prevent spills) at 4
C. The lid of the Falcon tube should
only be partially closed, for as the yeast ferments, carbon dioxide is produced, and the tube can burst if the gas cannot be
released.
8. Embryos can be collected throughout the day or overnight,
and selected under the stereomicroscope based on their morphology [33]; or collections can be timed and embryos can be
aged until they reach the appropriate developmental stage.
Development is 1.8 times slower at 18
C than at 25
C [34],
a fact that can be used to facilitate experiments. For example, to
select embryos at stage 14–15 of embryonic development for
wound healing assays, collect embryos for 2 h and age the plate
for 10 h at 25
C or for 18 h at 18
C. dsRNA injections must
be conducted in syncytial embryos, before cells form, and thus,
embryos must be collected for 30–60 min and immediately
prepared for injection.
9. Two strips of plasticine can be used as supports to facilitate
retrieval of the coverslip from the Petri dish (Fig. 1e).
10. To avoid desiccation, do not spend more than 5 min aligning
embryos. At the end, use the back of the forceps to remove all
the non-aligned embryos from the agar block so that they are
not transferred to the coverslip.
11. To image the ventral epidermis in an inverted microscope (e.g.,
for wound healing assays), orient embryos with their convex
(ventral) side up (Fig. 1f, left). To image dorsally (e.g., to
image dorsal closure or cardiac morphogenesis), the convex
side of the embryo should face the agar block (Fig. 1f, right).
12. If embryos burst when the oxygen-permeable membrane is
placed on top of the coverslip, or if the oil does not spread to
cover the entire coverslip, repeat using an additional drop of
oil. On the contrary, if embryos float and move in between the
membrane and the coverslip, reduce the amount of oil.
13. Dehydration times will be longer when humidity is high. Begin
with 5 min. If embryos are plump and the vitelline membrane is
hard to pierce with a microneedle, increase the dehydration
time in 1-min increments. If the vitelline membrane forms
folds or does not slightly resist deformation by the needle,
decrease the dehydration time.
14. For microinjections, it is often preferable to use only halocarbon oil 700 without mixing with halocarbon oil 27. The
greater viscosity of halocarbon oil 700 helps preserve the
integrity of the embryo and prevent “leaks” during the injection procedure.
214
Gordana Scepanovic et al.