This procedure begins with injection of a single cell zebra fish embryo positioned in an injection mold such that the cell is oriented facing away from the injection apparatus. The embryos are then injected with MRNA with the needle moving through the yolk and into the cell. The next day.
A plastic mounting slide is filled with aros. An anesthetized embryo is then placed inverted in a hole in the aros in imaged by confocal microscopy. Hi, I'm Grabham from the lab of Dr.Hazel Sieve at the Whitehead Institute for Biomedical Research and the biology department at MIT.
Today we will show you a procedure for injecting zebrafish embryos at the single cell stage and the subsequent live imaging of the brain by confocal microscopy. In our lab, we use this procedure to study vertebrate brain morphogenesis at single cell resolution. So let's get started.
The mRNA used in this procedure is transcribed from a plasmid encoding C-A-X-E-G-F-P-R-N-A-A membrane bound GFP First linearize the plasmid by digestion with not one, then transcribe membrane GFP mRNA from the linearized plasmid using the M message and machine kit after the mRNA has been transcribed, quantifying dilute the resulting mRNA and water to one microgram per one microliter, Eloqua, and store at negative 80 degrees Celsius until needed. On the day before injection set up mating cages, separating the males from the females on the day of injection. Use a setter instrument micropipet polar to pull capillary needles for injection.
Prepare an injection mold of 1%AGA rose with lanes the width of embryos at the one cell stage on the day of injection, thaw an aliquot of membrane GFP mRNA at a concentration of one microgram per microliter on ice. Dilute the mRNA one to five in water to a final concentration of 200 nanograms per microliter and keep on ice. Load the capillary needle with one microliter of prepared membrane, GFP mRNA at 200 nanograms per microliter.
Here we add 0.05%phenol red to the mRNA to make it visible for injections. Place the loaded needle into a micro manipulator attached to a gas powered micro injector. Adjust the injection volume to one nanoliter.
Next, pull the dividers on the wild type fish set up in mating cages from the previous day. Collect the embryos as soon as they are laid. Fill the AGA rose injection mold with embryo medium and orient the embryos in the mold with a single cell on the side away from the micro manipulator.
Inject through the corion and yolks such that the mRNA is deposited directly into the single cell. Inject approximately 50 embryos per experiment. However, if the cell has divided, do not inject after the embryos have been injected, incubate them at 28 degrees Celsius overnight in embryo medium.
After the overnight incubation, the injected embryos are ready for mounting and imaging. To begin this procedure, remove the corion from the embryos with forceps under a stereo microscope one hour before the time point of interest, prepare a slide for mounting up to four embryos. Then fill the slide with 0.7%aros just to the top of the slide and wait for about 20 minutes or until the aros solidifies.
Once the aros has solidified, make a small hole in the aeros using a 200 microliter pipette tip to form cylindrical holes in which to mount each embryo. Remove the aros plugs with forceps before mounting. Place the embryos on the aeros filled slide under a dissecting microscope.
Add 50 microliters of trica to anesthetize. The embryos. Use forceps to orient the embryos inverted in the cylindrical holes in the aros with the brain or region of interest against the underlying cover slip cover.
The embryos in the AGA rose with another cover slip and secure with silicone vacuum grease. The embryos are now ready for imaging. Image the embryo using an inverted fluorescent laser scanning confocal microscope as shown here, or spinning disc confocal microscope image at 63 x or higher to collect high resolution images of single cells within the neuro epithelium.
Images are exported as TIF files from the LSM software and analyzed using Photoshop. This is a representative confocal image of a 24 hour zebrafish embryo, neuro epithelium between the midbrain and hindbrain ventricles. M indicates the midbrain, ventricle, and H indicates the hindbrain ventricle.
Each cell is labeled with membrane bound GFPE. We've just demonstrated a technique that allows you to analyze the developing zebrafish brain as single cell resolution. This technique has enabled us to study a novel type of cell shape change called BA basal constriction.
It could also be used to analyze other types of phenomena and to significantly expand our understanding of vertebrae organogenesis, as well as its underlying cell biology. So that's it. Thanks for watching and good luck with your experiments.
