2.3 Microscopy
and Image Analysis
1. Upright confocal microscope with environmental chamber and
smaller stage chamber to limit dehydration.
2. Fiji software with plugin Correct 3d Drift [3, 4].
3. Optional: Imaris (Bitplane AG).
3 Methods
3.1 Prepare Culture
Dishes
1. The preparation of culture dishes may be performed up to
1 week in advance.
The critical steps are outlined here but can also be found in
[5]. Crack an egg into a large dish and use a 10 mL syringe with
18 g needle to extract thin albumen. Repeat until you have
collected 30 mL of thin albumen. Place in 49
C water bath or
similar heated incubator.
2. Dissolve 180 mg of Agar into 30 mL PBS, then reduce temperature to 49
C by placing in water bath. If this solution is
too hot, it will cook the egg albumen so allow enough time to
cool to 49
C.
3. Mix Agar solution and egg albumen, then add 600 μL penicillin/streptomycin and immediately pour into 60 mm petri
dishes and cover with lid. About 7 mL per dish (see Note 1).
4. After the medium has solidified, the culture dishes can be
stored at 4
C in a container with a wet paper towel for about
7 days before drying out or finding bacterial growth on
medium.
3.2 Label Embryos
1. Incubate embryos in humidified egg incubator to desired
developmental stage.
2. Add Penicillin–Streptomycin to ink and Ringer’s solution in a
small petri dish. Use a 1 mL syringe and 25G needle to hold
this solution. This will be injected underneath the embryo to
provide contrast to visualize.
3. Cut a window in the eggshell large enough to allow for visualization and microinjection of the embryo.
4. Inject the ink solution underneath embryo by inserting the
needle into the yolk outside of the area of the embryo.
5. Remove the vitelline membrane in the area above the embryo
that is to be fluorescently labeled using a tungsten needle or
fine forceps. Hydrate the embryo frequently by gently dripping
Ringers solution onto the vitelline membrane.
6. Inject DiI solution into neural tubes of embryos using picospritzer and glass needles. Take care to keep the DiI contained
within the neural tube since it will label all tissue it touches.
7. Reseal eggs with tape.
Live Imaging of EMT in Chick Neural Crest
109
and Image Analysis
1. Upright confocal microscope with environmental chamber and
smaller stage chamber to limit dehydration.
2. Fiji software with plugin Correct 3d Drift [3, 4].
3. Optional: Imaris (Bitplane AG).
3 Methods
3.1 Prepare Culture
Dishes
1. The preparation of culture dishes may be performed up to
1 week in advance.
The critical steps are outlined here but can also be found in
[5]. Crack an egg into a large dish and use a 10 mL syringe with
18 g needle to extract thin albumen. Repeat until you have
collected 30 mL of thin albumen. Place in 49
C water bath or
similar heated incubator.
2. Dissolve 180 mg of Agar into 30 mL PBS, then reduce temperature to 49
C by placing in water bath. If this solution is
too hot, it will cook the egg albumen so allow enough time to
cool to 49
C.
3. Mix Agar solution and egg albumen, then add 600 μL penicillin/streptomycin and immediately pour into 60 mm petri
dishes and cover with lid. About 7 mL per dish (see Note 1).
4. After the medium has solidified, the culture dishes can be
stored at 4
C in a container with a wet paper towel for about
7 days before drying out or finding bacterial growth on
medium.
3.2 Label Embryos
1. Incubate embryos in humidified egg incubator to desired
developmental stage.
2. Add Penicillin–Streptomycin to ink and Ringer’s solution in a
small petri dish. Use a 1 mL syringe and 25G needle to hold
this solution. This will be injected underneath the embryo to
provide contrast to visualize.
3. Cut a window in the eggshell large enough to allow for visualization and microinjection of the embryo.
4. Inject the ink solution underneath embryo by inserting the
needle into the yolk outside of the area of the embryo.
5. Remove the vitelline membrane in the area above the embryo
that is to be fluorescently labeled using a tungsten needle or
fine forceps. Hydrate the embryo frequently by gently dripping
Ringers solution onto the vitelline membrane.
6. Inject DiI solution into neural tubes of embryos using picospritzer and glass needles. Take care to keep the DiI contained
within the neural tube since it will label all tissue it touches.
7. Reseal eggs with tape.
Live Imaging of EMT in Chick Neural Crest
109