prepared, with the corner of the paper grabbed by the forceps.
Then dry on the regular paper towel. Repeat this with all
9 drops of water so the embryos are washed thoroughly. Keep
the damp paper towel with embryos in a lid of a 35 mm dish (see
Notes 2 and 3).
3.3 Embryo
Mounting
1. Clean the glass of the 35 mm glass bottom dish with Kimwipes
soaked in 70% ethanol. Do not touch the rim of the well to
prevent rubbing off debris.
2. Pick up each embryo one by one from the paper towel using
the paint brush with a single bristle. Land the embryos on the
glass in the dish. Avoid placing embryos on any remaining
debris on the glass (see Note 4).
3. After placing all embryos on the glass, put a few drops of water
on top of the embryos. Rock the dish so that the whole well is
covered by the water. Due to both the glass and vitelline
membrane being hydrophobic, embryos stick to the glass
underwater without floating. An air bubble will often form
attaching to each embryo.
4. Carefully pick up each embryo using the one-bristle paint
brush, within the water, leaving the air bubble on the glass.
Do not lift the embryo outside the water, otherwise the
embryo will be lost.
5. Choose a clean area, adjust the angles of the paint brush so that
the side of the embryo to be imaged is landed on the glass. To
image the mesoderm, have the ventral posterior side of the
embryo landing first on the glass. Gently lay the embryo
down to the middle of its ventral side on the glass. Try to
arrange the embryos in one or two rows to facilitate switching
between embryos with high magnification objective (see Notes 5
and 6).
6. Once all embryos are orientated correctly, cover the well of
water with a small piece of air permeable membrane. Place the
hydrophilic side of the membrane down and let the water
spread to the entire membrane. Let the excessive water run
away from the membrane so that the membrane sticks to the
petri dish (see Note 7).
3.4 Confocal
Imaging
1. Put a drop of immersion oil on the glass bottom of the dish
where the embryos are located on the other side of the glass.
Place the dish on the motorized stage of the confocal microscope (see Note 8).
2. Use the 63Â/1.4 oil objective (Plan Apochromatic). It can
cover about 10 Â 20 cell on the ventral side of the embryo.
Watch the embryos from above the stage and move them to the
center of the objective. Embryos can be seen as white spots by
Live Imaging of EMT in Chick Neural Crest
73
Then dry on the regular paper towel. Repeat this with all
9 drops of water so the embryos are washed thoroughly. Keep
the damp paper towel with embryos in a lid of a 35 mm dish (see
Notes 2 and 3).
3.3 Embryo
Mounting
1. Clean the glass of the 35 mm glass bottom dish with Kimwipes
soaked in 70% ethanol. Do not touch the rim of the well to
prevent rubbing off debris.
2. Pick up each embryo one by one from the paper towel using
the paint brush with a single bristle. Land the embryos on the
glass in the dish. Avoid placing embryos on any remaining
debris on the glass (see Note 4).
3. After placing all embryos on the glass, put a few drops of water
on top of the embryos. Rock the dish so that the whole well is
covered by the water. Due to both the glass and vitelline
membrane being hydrophobic, embryos stick to the glass
underwater without floating. An air bubble will often form
attaching to each embryo.
4. Carefully pick up each embryo using the one-bristle paint
brush, within the water, leaving the air bubble on the glass.
Do not lift the embryo outside the water, otherwise the
embryo will be lost.
5. Choose a clean area, adjust the angles of the paint brush so that
the side of the embryo to be imaged is landed on the glass. To
image the mesoderm, have the ventral posterior side of the
embryo landing first on the glass. Gently lay the embryo
down to the middle of its ventral side on the glass. Try to
arrange the embryos in one or two rows to facilitate switching
between embryos with high magnification objective (see Notes 5
and 6).
6. Once all embryos are orientated correctly, cover the well of
water with a small piece of air permeable membrane. Place the
hydrophilic side of the membrane down and let the water
spread to the entire membrane. Let the excessive water run
away from the membrane so that the membrane sticks to the
petri dish (see Note 7).
3.4 Confocal
Imaging
1. Put a drop of immersion oil on the glass bottom of the dish
where the embryos are located on the other side of the glass.
Place the dish on the motorized stage of the confocal microscope (see Note 8).
2. Use the 63Â/1.4 oil objective (Plan Apochromatic). It can
cover about 10 Â 20 cell on the ventral side of the embryo.
Watch the embryos from above the stage and move them to the
center of the objective. Embryos can be seen as white spots by
Live Imaging of EMT in Chick Neural Crest
73