3.2.1 Glue Method
1. Using a razor blade, cut out a rectangular piece of agar from a
clean apple juice agar plate. Leave the agar block on the covered
plate so that it does not dry out.
2. Using a glass pipette, place a drop of heptane glue on a coverslip. Tilt the coverslip so that the glue forms a thin layer along a
stripe on the surface of the coverslip. Store the coverslip glueside up on a Petri dish so that dirt does not stick to the glue (see
Note 9).
3. Retrieve the plate containing the embryos from the collection
cage and replace it with a new plate.
4. Pour 50% bleach in the plate containing the embryos and
incubate for 90–120 s. During the incubation, gently shake
the plate with circular movements to dislodge embryos from
the yeast and agar.
5. Pour the bleach and embryos into a wire mesh basket.
6. 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.
7. Using a clean, fine paper towel, dab the bottom and sides of the
basket to dry out any remaining bleach and water.
8. Place the agar block cut out in step 1 on a Petri dish cover
under the stereoscope (Fig. 1c).
9. Using a paintbrush, gently transfer embryos from the mesh
basket onto the agar block (Fig. 1c).
10. Working under the stereoscope, line up and orient the embryos
using a pair of forceps (see Notes 10 and 11). Epi-illumination
makes it easier to visualize embryonic morphology.
11. Transfer embryos onto the coverslip by gently pressing the
glue-painted coverslip surface against the agar block. For
embryo injection proceed to the next section without covering
the embryos in oil. If ready to image, use a glass pipette to
cover the embryos with three drops of halocarbon oil mixture,
tilting the coverslip as necessary to facilitate the spread of oil
(Fig. 1d, e).
3.2.2 Teflon Membrane
Method
1. Retrieve the plate containing the embryos from the collection
cage and replace it with a new plate.
2. Pour 50% bleach in the plate containing the embryos and
incubate for 90–120 s. During the incubation, gently shake
the plate with circular movements to dislodge embryos from
the yeast and agar.
3. Pour the bleach and embryos into a wire mesh basket.
Live Imaging in Drosophila Embryos
207
1. Using a razor blade, cut out a rectangular piece of agar from a
clean apple juice agar plate. Leave the agar block on the covered
plate so that it does not dry out.
2. Using a glass pipette, place a drop of heptane glue on a coverslip. Tilt the coverslip so that the glue forms a thin layer along a
stripe on the surface of the coverslip. Store the coverslip glueside up on a Petri dish so that dirt does not stick to the glue (see
Note 9).
3. Retrieve the plate containing the embryos from the collection
cage and replace it with a new plate.
4. Pour 50% bleach in the plate containing the embryos and
incubate for 90–120 s. During the incubation, gently shake
the plate with circular movements to dislodge embryos from
the yeast and agar.
5. Pour the bleach and embryos into a wire mesh basket.
6. 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.
7. Using a clean, fine paper towel, dab the bottom and sides of the
basket to dry out any remaining bleach and water.
8. Place the agar block cut out in step 1 on a Petri dish cover
under the stereoscope (Fig. 1c).
9. Using a paintbrush, gently transfer embryos from the mesh
basket onto the agar block (Fig. 1c).
10. Working under the stereoscope, line up and orient the embryos
using a pair of forceps (see Notes 10 and 11). Epi-illumination
makes it easier to visualize embryonic morphology.
11. Transfer embryos onto the coverslip by gently pressing the
glue-painted coverslip surface against the agar block. For
embryo injection proceed to the next section without covering
the embryos in oil. If ready to image, use a glass pipette to
cover the embryos with three drops of halocarbon oil mixture,
tilting the coverslip as necessary to facilitate the spread of oil
(Fig. 1d, e).
3.2.2 Teflon Membrane
Method
1. Retrieve the plate containing the embryos from the collection
cage and replace it with a new plate.
2. Pour 50% bleach in the plate containing the embryos and
incubate for 90–120 s. During the incubation, gently shake
the plate with circular movements to dislodge embryos from
the yeast and agar.
3. Pour the bleach and embryos into a wire mesh basket.
Live Imaging in Drosophila Embryos
207