4. Thoroughly wash embryos in the mesh basket with tap water
using a squirt bottle. Continue to wash embryos for 1–2 min.
Dab the bottom and sides of the basket with a fine paper towel
once during the process to ensure maximum bleach removal.
5. Using a clean fine paper towel, dab the bottom and sides of the
basket to dry out any remaining bleach and water.
6. Using a paintbrush, transfer embryos from the basket on to the
surface of the clean apple juice agar plate.
7. Using a glass pipette, cover embryos in halocarbon oil.
8. Add three drops of halocarbon oil on a coverslip using a glass
pipette.
9. Working under the stereoscope, use forceps (or a paintbrush)
to transfer embryos from the agar plate and into the oil drop on
the surface of the coverslip. Epi-illumination makes it easier to
visualize embryonic morphology.
10. Place the oxygen-permeable membrane on the coverslip, taking care to minimize embryo compression and bursting (see
Note 12).
3.3 Microinjection
1. Begin with embryos glued to a coverslip, but not covered in oil.
2. Dehydrate embryos by placing the coverslip with the glued
embryos in a sealed container full of silica beads for 5–15 min
(see Note 13).
3. Remove embryos from the container and cover them immediately with three drops of halocarbon oil mixture (see Note 14),
tilting the coverslip as necessary to spread of the oil.
4. Prepare a pulled microneedle (see Note 15) by loading 1–2 μL
of the substance to be injected (typically small molecule inhibitors, dyes, or dsRNA) into the intact needle.
5. Place the slide to break the microneedle on the microscope
stage, coverslip-side up. Add a drop of halocarbon oil on the
edge of the coverslip.
6. Carefully load the needle onto the pneumatic pump nozzle,
mounted on the micromanipulator (Fig. 2c
0 ).
7. Using the micromanipulator, carefully lower the needle toward
the slide until the tip of the needle is inside the drop of oil at the
edge of the coverslip. Focus the microscope on the needle tip.
Adjust the position of the needle using the micromanipulator
until both the needle tip and the edge of the coverslip are on
the same plane.
8. Try to eject liquid with the pump to make sure that the needle
is not broken. No bubbles should come out of the tip of the
needle.
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Gordana Scepanovic et al.
using a squirt bottle. Continue to wash embryos for 1–2 min.
Dab the bottom and sides of the basket with a fine paper towel
once during the process to ensure maximum bleach removal.
5. Using a clean fine paper towel, dab the bottom and sides of the
basket to dry out any remaining bleach and water.
6. Using a paintbrush, transfer embryos from the basket on to the
surface of the clean apple juice agar plate.
7. Using a glass pipette, cover embryos in halocarbon oil.
8. Add three drops of halocarbon oil on a coverslip using a glass
pipette.
9. Working under the stereoscope, use forceps (or a paintbrush)
to transfer embryos from the agar plate and into the oil drop on
the surface of the coverslip. Epi-illumination makes it easier to
visualize embryonic morphology.
10. Place the oxygen-permeable membrane on the coverslip, taking care to minimize embryo compression and bursting (see
Note 12).
3.3 Microinjection
1. Begin with embryos glued to a coverslip, but not covered in oil.
2. Dehydrate embryos by placing the coverslip with the glued
embryos in a sealed container full of silica beads for 5–15 min
(see Note 13).
3. Remove embryos from the container and cover them immediately with three drops of halocarbon oil mixture (see Note 14),
tilting the coverslip as necessary to spread of the oil.
4. Prepare a pulled microneedle (see Note 15) by loading 1–2 μL
of the substance to be injected (typically small molecule inhibitors, dyes, or dsRNA) into the intact needle.
5. Place the slide to break the microneedle on the microscope
stage, coverslip-side up. Add a drop of halocarbon oil on the
edge of the coverslip.
6. Carefully load the needle onto the pneumatic pump nozzle,
mounted on the micromanipulator (Fig. 2c
0 ).
7. Using the micromanipulator, carefully lower the needle toward
the slide until the tip of the needle is inside the drop of oil at the
edge of the coverslip. Focus the microscope on the needle tip.
Adjust the position of the needle using the micromanipulator
until both the needle tip and the edge of the coverslip are on
the same plane.
8. Try to eject liquid with the pump to make sure that the needle
is not broken. No bubbles should come out of the tip of the
needle.
208
Gordana Scepanovic et al.