first channel you will need to say number of images 300, starting image 1, increment 30 and sort numerically. This will
generate a stack of the first channel. For the second channel
would be number of images 300, starting image 301, increment
30 and sort numerically; and so on.
7. There are a number of variables that can cause the Registration
to fail. If the new stack has not been corrected properly (e.g.,
has black frames or incorrect rotations), try one or both of
these steps:
(a) Check the focused stack created in step 4 and make sure
that all frames display the anatomical features in focus,
without drastic changes between consecutive frames.
(b) Adjust the brightness of the stack “Image > Adjust >
Brightness/Contrast” to make the landmarks more
evident.
8. If you are not sure about the calibration of your instrument,
you can calibrate it by taking an image of a graded stage reticule
and calculate the XY length of a pixel. Use the smallest reticule
you can find, a micrometre scale 10 mm in 0.1 mm division is
suitable, take an image. Using the measuring tool from the
microscope software, measure the number of pixels each reticule unit spans. Or open your reticule image in Fiji, draw
horizontal straight line spanning one reticule unit using the
line tool, and pressing shift as you draw. Press “m” the number
of pixels will be under length in the Results table. Calculate the
pixel length in μm.
9. Gal4ER fusion was generated using the Kalt4 form of the Gal4
activator [10] and the extracellular domain of the human oestrogen receptor.
10. Plasmid DNA of UAS-driven constructs with or without transposable elements (Tol2 [13] or ACDS [14]) has been successfully expressed after injection. We have generated double UAS
vectors driving the expression of super folded GFP in one
strand and any protein of interest in the other. In our hands,
super folded GFP and Scarlett are the brightest green and red
fluorescent proteins, respectively. The use of the zebrafish
codon sequence has a major impact in the expression levels in
both cases. In all cases, 20 ng of plasmid DNA is injected.
Transposase mRNA is injected when required; the concentration of RNA to inject needs to be experimentally defined as is
batch dependent. It is very important to inject into the cell of
early one-cell stage embryos, when the cell is still flat surrounding the yolk. The number of clones obtained is drastically
reduced if embryos are injected once the first cell has
plumped up.
104
Zain Alhashem et al.
generate a stack of the first channel. For the second channel
would be number of images 300, starting image 301, increment
30 and sort numerically; and so on.
7. There are a number of variables that can cause the Registration
to fail. If the new stack has not been corrected properly (e.g.,
has black frames or incorrect rotations), try one or both of
these steps:
(a) Check the focused stack created in step 4 and make sure
that all frames display the anatomical features in focus,
without drastic changes between consecutive frames.
(b) Adjust the brightness of the stack “Image > Adjust >
Brightness/Contrast” to make the landmarks more
evident.
8. If you are not sure about the calibration of your instrument,
you can calibrate it by taking an image of a graded stage reticule
and calculate the XY length of a pixel. Use the smallest reticule
you can find, a micrometre scale 10 mm in 0.1 mm division is
suitable, take an image. Using the measuring tool from the
microscope software, measure the number of pixels each reticule unit spans. Or open your reticule image in Fiji, draw
horizontal straight line spanning one reticule unit using the
line tool, and pressing shift as you draw. Press “m” the number
of pixels will be under length in the Results table. Calculate the
pixel length in μm.
9. Gal4ER fusion was generated using the Kalt4 form of the Gal4
activator [10] and the extracellular domain of the human oestrogen receptor.
10. Plasmid DNA of UAS-driven constructs with or without transposable elements (Tol2 [13] or ACDS [14]) has been successfully expressed after injection. We have generated double UAS
vectors driving the expression of super folded GFP in one
strand and any protein of interest in the other. In our hands,
super folded GFP and Scarlett are the brightest green and red
fluorescent proteins, respectively. The use of the zebrafish
codon sequence has a major impact in the expression levels in
both cases. In all cases, 20 ng of plasmid DNA is injected.
Transposase mRNA is injected when required; the concentration of RNA to inject needs to be experimentally defined as is
batch dependent. It is very important to inject into the cell of
early one-cell stage embryos, when the cell is still flat surrounding the yolk. The number of clones obtained is drastically
reduced if embryos are injected once the first cell has
plumped up.
104
Zain Alhashem et al.