This procedure begins with the staging of zebrafish embryos. When the embryos have reached the stage of interest, remove them from the corion. Next holes are created in an aros coated dish with a pipette tip for mounting the embryos and the embryos are placed gently in the holes with their tails down.
After the embryos are mounted, a fluorescent die is carefully injected into the ventricle space of the embryo through the hind brain. After ventricle injection, the embryos are imaged and the data is processed using Photoshop. Hi, I'm Jennifer Gutman from the laboratory of Dr.Hazel Sieve at the Whitehead Institute for Biomedical Research in MIT in Cambridge, Massachusetts.
Today we will show you a procedure for zebrafish brain ventricle injection. We use this procedure in our laboratory to study brain ventricle formation and neuro epithelial morphogenesis in live zebrafish. Due to the nature of transparent zebrafish embryos, characterizing the brain ventricle shape can be difficult.
This technique is a useful and simple way to differentiate the fluid-filled space from the surrounding tissue and to characterize mutant embryos that may have brain ventricle formation defects. So let's get started. To prepare for injecting the zebrafish brain ventricle, we start by making the injection needles.
A Sutter instrument's needle polar is used to pull the capillary needles. The capillary needle is then filled with a fluorescent dye, such as Texas red dextrin. Next, the filled needle is mounted in a micro manipulator in microinjection apparatus.
Cut the needle to the appropriate size at an angle. To create a beveled tip, measure the drop size of the needle and check that it is between one to two nanoliters per injection in oil. To begin this procedure stage, the embryos, according to Kimmel at all embryos should be injected 30 minutes earlier than the stage of interest.
For example, to study the ventricles at 24 hours post fertilization inject the embryos at 23 and a half hours post fertilization. The embryo at the stage of interest is placed under the stereo microscope, and the corion is carefully removed from the embryo using forceps. Next, an aros dish for the embryos is made using a plastic dish coated with 1%aros poke holes in the aros with a plastic one to 200 microliter pipet tip.
Carefully remove the aros plugs from the holes using forceps. Transfer the embryo and embryo media into the aros dish with holes. Add trica to the media to anesthetize the embryo and prevent movement.
During the experiment, gently place the embryo tail down into the hole and orient it so the injection apparatus is on the posterior side of the embryo. Now that the embryo is placed and oriented correctly in the arose dish, we are ready for the brain ventricle injection. First, arrange the micro-manipulation setup so that the needle tip is in the same field of view as the embryo on high power.
Carefully put the needle through the thin roof plate of the hind brain just posterior to the R zero R one hinge point without hitting the brain tissue below, inject just enough dye to completely fill the ventricles. It may take several injections to fill the ventricle space depending on the stage of the embryo. When the embryo has been injected and is ready for imaging, a new aros dish for the embryo is prepared.
Using a clean 1%aros coated dish, poke holes in the dish and remove the plugs. Transfer the embryo to the dish and add trica to anesthetize the embryo and prevent movement. Place the tail of the ventricle injected embryo into the hole in position for a dorsal image.
The embryo is now ready for imaging. Take an image using transmitted light. It is very critical to not move the embryo or the microscope.
Next, change the settings on the microscope and take an image with fluorescent light. Reposition the embryo to take lateral images with both transmitted light and fluorescent light. Save all the images as to files for image processing of Photoshop.
To process the images in Photoshop, open the transmitted light and fluorescent light images of the same embryo taken in the same position. Be sure all of the settings are the same For each image, drag your fluorescent image onto the transmitted light image and line them up exactly with the fluorescent image selected. Select image adjustments, and then replace color and change the black background to white.
In the replace color window, adjust the fuzziness factor to the right until the fluorescent image looks uniform in color. In the layers window, select multiply. To view the overlay images, your ventricle space image should match the transmitted light image.Exactly.
Save this file and repeat for any other images. If the ventricle injection is performed correctly, the images should have sharp edges in non diffuse dye as shown here in these images of 25 hours. Post fertilization while type embryos panel A shows the bright field image, panel B shows the fluorescent image and panel C shows the overlay image.
A lateral view of the overlay image is shown in Panel D.On the other hand, if during the ventricle injection the needle is inserted too far, it'll enter the brain tissue below the ventricle space and lead to an incorrect injection. This results in dye that is visible outside of the ventricle space and in the yolk as shown here. Panel E shows the bright field image.
Panel F shows the fluorescent image and panel G shows the overlaid image. A lateral view of the overlaid image is shown in panel H.The arrows indicate regions where the dye has gone beyond the brain ventricle space. In this video, we demonstrated how to inject fluorescent dye into developing zebrafish brain ventricles.
This method is used to visualize the brain ventricle space in contrast to the surrounding neuro epithelium, and is extremely useful for determining the shape of the ventricle space as well as the shape of the surrounding brain tissue. This technique allows us to better understand the process of brain ventricle formation and brain morphogenesis over time in the live embryo. It's an excellent tool for studying brain ventricle formation defects, and for initial characterization of brain morphogenesis mutants.
So that's it. Thanks for watching and good luck with your experiments.
