The overall goal of this procedure is to quantify permeability of the zebrafish, embryonic, neuro epithelium. This is accomplished by first injecting a 70 kilodalton fitzy dextran into the brain ventricles. Next, a time course of brightfield and fluorescent images is acquired.
Then the distance of the fluorescent die from the forebrain hinge point at each time point is measured. Finally, the distance traveled over time is calculated. Ultimately, results can be obtained that show changes in neuro epithelial permeability by comparison of different genetic backgrounds or environmental conditions.
The overall goal of this procedure is to quantify permeability in the zebrafish embryonic neuro epithelium. To prepare for injection load a previously prepared injection needle with fluorescent dye, mount the needle on the manipulator and microinjection apparatus. Use forceps to carefully break the microinjection needle to roughly two micron in width.
Measure the drop size in oil, adjusting the injection time and pressure so that each injection delivers one nanoliter To prepare the embryos coat two dishes with 1%agros in water for each condition, and using a 200 microliter pipette tip, poke holes into the agros and remove the plugs. Fill the dishes with embryo medium under a stereo microscope. Use forceps to chlorinate embryos, which are 18 hours post fertilization or older.
Then transfer the embryos into one of the agros coated dishes to anesthetize embryos. Add trica to the dish and wait until the embryos stop moving. Orient the embryos to look at their dorsal side by putting the tail of the embryo into the hole.
If your micro manipulator is on the right, then move the embryo so that the forebrain is to the left and hind brain is on the right. Position the needle at the widest point of the hindbrain ventricle. Next, carefully pierce the roof plate of the hind brainin ventricle, being sure not to go through the depth of the brain into the yolk.
Then inject one to two nanoliters of fluorescent dye into the ventricles, making sure the dye fills the whole length of the brain.Ventricles. Transfer the embryos to the second agros dish filled with embryo, medium, and re anesthetize them. Move immediately to imaging to obtain time.
Zero images to image the embryos. Orient them with their tails in the hole as before, under a dissecting microscope that has both transmitted and fluorescent light. Take a bright field dorsal image.
Maintain a constant magnification between embryos to allow for direct comparison without moving the embryo microscope or dish. Take a corresponding fluorescent image, repeat for each embryo and each time point desired. To quantify the dye movement, use Photoshop to merge the brightfield and fluorescent images.
Then to measure the distance the dye front moves, use image J to open the merge to file and use the line tool to draw a line from the forebrain hinge point to the die front at a 10 to 20 degree angle from the neuro epithelium, select the measurement tool to calculate the length of the line. Finally, calculate the net distance. The D front moved over time by subtracting the distance at time zero from other time points.
Plot the movements on a graph shown. Here is an example of neuro epithelial permeability results using wild type embryos. The merged brightfield images shown here were taken at 22 and 24 hours post fertilization respectively.
The white line indicates the distance of the D front from the forebrain ventricle. The graph shows hypothetical sample permeability data demonstrating the ability to determine an increase in permeability shown in green, or a decrease in permeability shown in red compared to control embryos in blue. To accurately differentiate permeability, it is useful to test dyes with different molecular weights to identify a size that is only slightly leaky in wild type or control embryos.
This allows for identification of genetic mutants or environmental conditions that either increase or decrease permeability for the 24 hours. Post fertilization, wild type zebrafish neuro epithelium. 70 kilodalton fitzy dextran leaks slowly over two hours, whereas 2000 kilodalton does not, and 10 kilodalton almost immediately leaks out.
Therefore, 70 kilodaltons is the ideal molecular weight to identify conditions that both increase and decrease neuro epithelial permeability. After watching this video, you should have a good understanding for how to quantify neuro epithelial permeability in the embryonic zebrafish.
