Remove the extra water with a p200 pipettor or a narrowtipped transfer pipette.
4. Yolk particles. Yolk is widely autofluorescent and will obscure a
bright-field image. If a considerable amount of yolk is above
the embryo, imaging will be more difficult. If gently washing
the embryo with fresh Ringer’s solution in the imaging dish
cannot be accomplished, place the embryo into a fresh dish of
Ringer’s solution and gently blow the yolk off with a transfer
pipette. The embryo can then be placed back on an EC culture
dish. Transferring the embryo should be avoided so the tissue
does not fold and the embryo stays warm. Small amounts of
yolk beneath the embryo may not be in the region of interest
and can be left alone.
5. Excess water. If there is too much water underneath the
embryo, it will slide in the culture dish during transfer to the
microscope and during imaging. If there is enough water
around the embryo to collect with a pipette, do so using either
a p200 pipettor or a narrow-tipped transfer pipette. Do not pull
on tissue, instead pull from outside the paper ring even tipping
the dish slightly while collecting the water.
6. Mounting multiple embryos. 35 mm dishes can also be used
for smaller embryos (
mounted in one 60 mm petri dish. It is easier to accomplish this
with younger embryos since they are smaller and thinner so the
possibility of touching the microscope objective to the embryo
is lessened. A small amount of vacuum grease can be used to
stabilize the dish to the stage for multi-position imaging but
may not be necessary.
References
1. Kulesa PM, Gammill LS (2010) Neural crest
migration: patterns, phases and signals. Dev
Biol 344(2):566–568. https://doi.org/10.
1016/j.ydbio.2010.05.005
2. McKinney MC, Fukatsu K, Morrison J,
McLennan R, Bronner ME, Kulesa PM (2013)
Evidence for dynamic rearrangements but lack
of fate or position restrictions in premigratory
avian trunk neural crest. Development 140
(4):820–830. https://doi.org/10.1242/dev.
083725
3. Preibisch S, Saalfeld S, Schindelin J, Tomancak P
(2010) Software for bead-based registration of
selective plane illumination microscopy data.
Nat Methods 7(6):418–419. https://doi.org/
10.1038/nmeth0610-418
4. Schindelin J, Arganda-Carreras I, Frise E,
Kaynig V, Longair M, Pietzsch T, Preibisch S,
Rueden C, Saalfeld S, Schmid B, Tinevez JY,
White DJ, Hartenstein V, Eliceiri K,
Tomancak P, Cardona A (2012) Fiji: an opensource platform for biological-image analysis.
Nat Methods 9(7):676–682. https://doi.org/
10.1038/nmeth.2019
5. Chapman SC, Collignon J, Schoenwolf GC,
Lumsden A (2001) Improved method for chick
whole-embryo culture using a filter paper carrier.
Dev Dyn 220(3):284–289. https://doi.org/10.
1002/1097-0177(20010301)220:3
6. McLennan R, Kulesa PM (2007) In vivo analysis
reveals a critical role for neuropilin-1 in cranial
neural crest cell migration in chick. Dev Biol 301
(1):227–239.
https://doi.org/10.1016/j.
ydbio.2006.08.019
114
Mary Cathleen McKinney and Paul M. Kulesa
4. Yolk particles. Yolk is widely autofluorescent and will obscure a
bright-field image. If a considerable amount of yolk is above
the embryo, imaging will be more difficult. If gently washing
the embryo with fresh Ringer’s solution in the imaging dish
cannot be accomplished, place the embryo into a fresh dish of
Ringer’s solution and gently blow the yolk off with a transfer
pipette. The embryo can then be placed back on an EC culture
dish. Transferring the embryo should be avoided so the tissue
does not fold and the embryo stays warm. Small amounts of
yolk beneath the embryo may not be in the region of interest
and can be left alone.
5. Excess water. If there is too much water underneath the
embryo, it will slide in the culture dish during transfer to the
microscope and during imaging. If there is enough water
around the embryo to collect with a pipette, do so using either
a p200 pipettor or a narrow-tipped transfer pipette. Do not pull
on tissue, instead pull from outside the paper ring even tipping
the dish slightly while collecting the water.
6. Mounting multiple embryos. 35 mm dishes can also be used
for smaller embryos (
mounted in one 60 mm petri dish. It is easier to accomplish this
with younger embryos since they are smaller and thinner so the
possibility of touching the microscope objective to the embryo
is lessened. A small amount of vacuum grease can be used to
stabilize the dish to the stage for multi-position imaging but
may not be necessary.
References
1. Kulesa PM, Gammill LS (2010) Neural crest
migration: patterns, phases and signals. Dev
Biol 344(2):566–568. https://doi.org/10.
1016/j.ydbio.2010.05.005
2. McKinney MC, Fukatsu K, Morrison J,
McLennan R, Bronner ME, Kulesa PM (2013)
Evidence for dynamic rearrangements but lack
of fate or position restrictions in premigratory
avian trunk neural crest. Development 140
(4):820–830. https://doi.org/10.1242/dev.
083725
3. Preibisch S, Saalfeld S, Schindelin J, Tomancak P
(2010) Software for bead-based registration of
selective plane illumination microscopy data.
Nat Methods 7(6):418–419. https://doi.org/
10.1038/nmeth0610-418
4. Schindelin J, Arganda-Carreras I, Frise E,
Kaynig V, Longair M, Pietzsch T, Preibisch S,
Rueden C, Saalfeld S, Schmid B, Tinevez JY,
White DJ, Hartenstein V, Eliceiri K,
Tomancak P, Cardona A (2012) Fiji: an opensource platform for biological-image analysis.
Nat Methods 9(7):676–682. https://doi.org/
10.1038/nmeth.2019
5. Chapman SC, Collignon J, Schoenwolf GC,
Lumsden A (2001) Improved method for chick
whole-embryo culture using a filter paper carrier.
Dev Dyn 220(3):284–289. https://doi.org/10.
1002/1097-0177(20010301)220:3
6. McLennan R, Kulesa PM (2007) In vivo analysis
reveals a critical role for neuropilin-1 in cranial
neural crest cell migration in chick. Dev Biol 301
(1):227–239.
https://doi.org/10.1016/j.
ydbio.2006.08.019
114
Mary Cathleen McKinney and Paul M. Kulesa