5. If time-lapse movies are required, add 0.5 μM tamoxifen to the
agarose mould, LMP and medium indicated in the imaging
protocol described.
3.7 Photo-Labelling
of Individual Neural
Crest Cells
1. Obtain Sox10:Dendra2
kg329Tg embryos and incubate at
28.5
C or 19
C (see above) until the desired stage. Heteroand homozygous embryos are bright enough to carry out the
experiment, but homozygous embryos are easier to work with
as the fluorescence intensity is higher.
2. At the desired developmental stage, dechorionate and transfer
to E3/tricaine medium to mount for photoconversion.
3. Add 0.1 mL of tricaine to 0.9 mL of 3% methyl cellulose in a
1.5 mL tube, vortex and let stand for 10 min for bubbles to
come up. With a plastic pipette transfer the embryos with the
absolute minimum amount of medium into the tube.
(a) If using an upright microscope, embryos can be placed
and oriented in an agarose mould as explained before (see
Subheading 3.1), or simply mounted in methyl cellulose:
add a thin layer (0.3–0.5 mL) of methyl cellulose to a
plastic Petri dish where the embryos will be located.
Transfer the embryos from the 1.5 mL tube into the
drop and orient in a line next to each other using a hair
knife.
(b) If using an inverted microscope mount in a drop of methyl
cellulose as explained but use a glass bottom dish; in this
case, make sure gently push in the embryos so they touch
the glass.
4. Put the embryo dish on the microscope stage and focus on the
embryos on the desired area. Take a quick image with 488 nm
illumination as reference image. Limit the exposure to the
488 nm laser to the minimum as it can photoconvert Dendra2 at high power.
5. Run a trial experiment to determine exposure time and power
to efficiently photoconvert Dendra2 using the 405 nm laser.
Start with minimum laser power and exposure times. Dendra2
is very sensitive and will totally bleach easily (green and red
emission).
6. Once the photoconversion conditions have been established,
select regions of interest around single or group of nuclei
required (Fig. 4). Make sure the region of interest selected is
as small as possible to avoid photoconversion of adjacent cells
(see Note 12).
7. After photoconversion, take a quick image using the 561 nm
laser to check that that desired cells have been photoconverted
successfully.
Tools to Study Neural Crest Migration in Zebrafish
101
agarose mould, LMP and medium indicated in the imaging
protocol described.
3.7 Photo-Labelling
of Individual Neural
Crest Cells
1. Obtain Sox10:Dendra2
kg329Tg embryos and incubate at
28.5
C or 19
C (see above) until the desired stage. Heteroand homozygous embryos are bright enough to carry out the
experiment, but homozygous embryos are easier to work with
as the fluorescence intensity is higher.
2. At the desired developmental stage, dechorionate and transfer
to E3/tricaine medium to mount for photoconversion.
3. Add 0.1 mL of tricaine to 0.9 mL of 3% methyl cellulose in a
1.5 mL tube, vortex and let stand for 10 min for bubbles to
come up. With a plastic pipette transfer the embryos with the
absolute minimum amount of medium into the tube.
(a) If using an upright microscope, embryos can be placed
and oriented in an agarose mould as explained before (see
Subheading 3.1), or simply mounted in methyl cellulose:
add a thin layer (0.3–0.5 mL) of methyl cellulose to a
plastic Petri dish where the embryos will be located.
Transfer the embryos from the 1.5 mL tube into the
drop and orient in a line next to each other using a hair
knife.
(b) If using an inverted microscope mount in a drop of methyl
cellulose as explained but use a glass bottom dish; in this
case, make sure gently push in the embryos so they touch
the glass.
4. Put the embryo dish on the microscope stage and focus on the
embryos on the desired area. Take a quick image with 488 nm
illumination as reference image. Limit the exposure to the
488 nm laser to the minimum as it can photoconvert Dendra2 at high power.
5. Run a trial experiment to determine exposure time and power
to efficiently photoconvert Dendra2 using the 405 nm laser.
Start with minimum laser power and exposure times. Dendra2
is very sensitive and will totally bleach easily (green and red
emission).
6. Once the photoconversion conditions have been established,
select regions of interest around single or group of nuclei
required (Fig. 4). Make sure the region of interest selected is
as small as possible to avoid photoconversion of adjacent cells
(see Note 12).
7. After photoconversion, take a quick image using the 561 nm
laser to check that that desired cells have been photoconverted
successfully.
Tools to Study Neural Crest Migration in Zebrafish
101