19. Open “Manual Tracking” plugin “Plugins > Tracking > Manual Tracking.”
20. On the “Tracking” window, click “Load Previous Track File.”
Fiji will ask you if you are sure that you want to load an existing
file; say “Yes.”
21. Select the text file you created previously, that will be uploaded
in a new data window.
22. At this point you can select how to overlay your tracks onto the
movie in the “Drawing” section of the Tracking window. The
top row (“Dots” / “Progressive Lines” / “Dots & Lines”) will
create a separate movie only with the tracks over a black background. The bottom row (“Overlay Dots” / “Overlay Lines”
/ “Overlay Dots & Lines”) will create a separate movie with
the tracks over the projection of your movie. “Dots” overlays a
dot on top of each tracked cell; “Lines” overlays the track,
progressing through time; “Dots & Lines” does both at the
same time.
23. You can now save this new file as a separate movie.
3.6 Time-Controlled
Induction of Protein
Expression
in Neural Crest
Transgenic embryos from the Sox10:Kalt4ER
kg328Tg line express
the Gal4ER fusion protein in all NC, which upon addition of
tamoxifen will move to the nucleus and initiate transcription of
any UAS-driven transgenes. This system allows protein overexpression only in NC at any required time of development.
1. Cross Sox10:Kalt4ER
kg328Tg transgenic fish to the desired
UAS regulated line for expression in the entire neural crest
population (see Note 9). Alternatively, for clonal induction,
inject UAS-driven plasmid DNA into one-cell stage embryos
(see Note 10).
2. Incubate the embryos at 28.5
C until the required stage.
Alternatively, to obtain stages from 11hpf onwards in the
morning, maintain embryos at 28.5
C until 50% epiboly,
then transfer them to 19
C overnight (see Note 11).
3. Embryos will be about 10hpf at 9:00 next morning. Move
them to 28.5
C until the desired stage.
4. Add tamoxifen to the E3 medium to a final concentration of
0.5 μM. Measure the necessary amount of E3 in a 50 mL
Falcon tube; add the embryos to be treated in their chorions
and set the volume correctly; dilute the 5 mM stock tamoxifen
10,000 times. Pour in to a desired Petri dish and incubate at
28.5
C until the desired stage. Expression of UAS-driven
proteins is observed after 30 min of tamoxifen addition and is
maximal after 1 h (Fig. 4).
100
Zain Alhashem et al.
20. On the “Tracking” window, click “Load Previous Track File.”
Fiji will ask you if you are sure that you want to load an existing
file; say “Yes.”
21. Select the text file you created previously, that will be uploaded
in a new data window.
22. At this point you can select how to overlay your tracks onto the
movie in the “Drawing” section of the Tracking window. The
top row (“Dots” / “Progressive Lines” / “Dots & Lines”) will
create a separate movie only with the tracks over a black background. The bottom row (“Overlay Dots” / “Overlay Lines”
/ “Overlay Dots & Lines”) will create a separate movie with
the tracks over the projection of your movie. “Dots” overlays a
dot on top of each tracked cell; “Lines” overlays the track,
progressing through time; “Dots & Lines” does both at the
same time.
23. You can now save this new file as a separate movie.
3.6 Time-Controlled
Induction of Protein
Expression
in Neural Crest
Transgenic embryos from the Sox10:Kalt4ER
kg328Tg line express
the Gal4ER fusion protein in all NC, which upon addition of
tamoxifen will move to the nucleus and initiate transcription of
any UAS-driven transgenes. This system allows protein overexpression only in NC at any required time of development.
1. Cross Sox10:Kalt4ER
kg328Tg transgenic fish to the desired
UAS regulated line for expression in the entire neural crest
population (see Note 9). Alternatively, for clonal induction,
inject UAS-driven plasmid DNA into one-cell stage embryos
(see Note 10).
2. Incubate the embryos at 28.5
C until the required stage.
Alternatively, to obtain stages from 11hpf onwards in the
morning, maintain embryos at 28.5
C until 50% epiboly,
then transfer them to 19
C overnight (see Note 11).
3. Embryos will be about 10hpf at 9:00 next morning. Move
them to 28.5
C until the desired stage.
4. Add tamoxifen to the E3 medium to a final concentration of
0.5 μM. Measure the necessary amount of E3 in a 50 mL
Falcon tube; add the embryos to be treated in their chorions
and set the volume correctly; dilute the 5 mM stock tamoxifen
10,000 times. Pour in to a desired Petri dish and incubate at
28.5
C until the desired stage. Expression of UAS-driven
proteins is observed after 30 min of tamoxifen addition and is
maximal after 1 h (Fig. 4).
100
Zain Alhashem et al.