2 Materials
2.1 Embryo
Collection
1. Plastic cups with punctured small holes.
2. 60 mm apple juice agar plates (to make 500 ml: Drosophila
Agar type II 9 g, sucrose 12 g, apple juice 250 ml, H 2 O
250 ml).
3. Yeast paste.
4. 60 mm petri dish lid.
2.2 Embryo
Preparation
and Mounting
1. Stereomicroscope with a transmitted light source (e.g., Zeiss
Stemi 508).
2. 200 μm pipettor or glass Pasteur pipette.
3. Fine forceps.
4. Paint brushes: one with most bristles removed and another one
with only one bristle.
5. 35 mm glass bottom Petri dish.
6. Absorbent C-fold paper towels: regular size and cut into about
2 cm  2 cm pieces.
7. Air permeable membrane.
8. Water in a wash bottle.
9. Halocarbon oil 27.
10. 4% sodium hypochlorite/bleach: dilute household bleach with
water (household bleach from grocery stores usually contains
6% or 8% sodium hypochlorite).
2.3 Live Imaging
1. Laser scanning confocal microscope.
2. Plan Apochromatic 63Â oil immersion objective.
3 Methods
3.1 Embryo
Collection
1. Place 40 to 60 young flies expressing desired fluorescent proteins in the egg collection cup and cover the opening with an
apple juice plate with a drop of yeast paste. Keep the cup upside
down with a rubber band securing the plate to the cup. Change
the plate every day to feed the flies for about 2 days so the flies
start to lay abundant amounts of eggs.
2. On the day of imaging, change to a room-temperature apple
juice plate with yeast paste on it and collect the eggs for desired
length of time. Keep the cup in a dark and undisturbed place to
increase egg laying. To image embryos from early cellularization to gastrulation, collect eggs for at least 2 h. As flies lay
more eggs at this temperature, keeping the egg collection cup
Live Imaging of EMT in Chick Neural Crest
71
2.1 Embryo
Collection
1. Plastic cups with punctured small holes.
2. 60 mm apple juice agar plates (to make 500 ml: Drosophila
Agar type II 9 g, sucrose 12 g, apple juice 250 ml, H 2 O
250 ml).
3. Yeast paste.
4. 60 mm petri dish lid.
2.2 Embryo
Preparation
and Mounting
1. Stereomicroscope with a transmitted light source (e.g., Zeiss
Stemi 508).
2. 200 μm pipettor or glass Pasteur pipette.
3. Fine forceps.
4. Paint brushes: one with most bristles removed and another one
with only one bristle.
5. 35 mm glass bottom Petri dish.
6. Absorbent C-fold paper towels: regular size and cut into about
2 cm  2 cm pieces.
7. Air permeable membrane.
8. Water in a wash bottle.
9. Halocarbon oil 27.
10. 4% sodium hypochlorite/bleach: dilute household bleach with
water (household bleach from grocery stores usually contains
6% or 8% sodium hypochlorite).
2.3 Live Imaging
1. Laser scanning confocal microscope.
2. Plan Apochromatic 63Â oil immersion objective.
3 Methods
3.1 Embryo
Collection
1. Place 40 to 60 young flies expressing desired fluorescent proteins in the egg collection cup and cover the opening with an
apple juice plate with a drop of yeast paste. Keep the cup upside
down with a rubber band securing the plate to the cup. Change
the plate every day to feed the flies for about 2 days so the flies
start to lay abundant amounts of eggs.
2. On the day of imaging, change to a room-temperature apple
juice plate with yeast paste on it and collect the eggs for desired
length of time. Keep the cup in a dark and undisturbed place to
increase egg laying. To image embryos from early cellularization to gastrulation, collect eggs for at least 2 h. As flies lay
more eggs at this temperature, keeping the egg collection cup
Live Imaging of EMT in Chick Neural Crest
71