急性腎障害を研究するためにゼブラフィッシュ幼生の静脈内Microinjections

The overall goal of this procedure is to create a model of acute kidney injury in zebrafish using the AMG Glycoside Gentamicin as the nephrotoxin. This is accomplished by first injecting gentamicin into the common cardinal vein of two days post fertilization, zebrafish larvae. The second step of the procedure is to score for emus phenotypes in four days post fertilization injected larvae.

The third step of the procedure is to process four days post fertilization zebrafish for immunohistochemistry staining with an antibody for kidney tubule epithelium am. The final step of the procedure is to section zebrafish, larvae and visualize for damaged tubules due to loss of cell polarity and disruption of tubule structure. Ultimately, results can be obtained that show damaged renal tubule segments through immunohistochemistry with a kidney epithelium antibody.

Hi, I am Carala San Cosentino from Neil Hari Laboratory in the Department of Developmental Biology at the University of Pittsburgh. Today we will show you a procedure to micro inject the amino glycoside Gentamycin into two days post fertilization zebrafish larva. We use this procedure in our laboratory to induce acute kidney injury in larva zebrafish.

So let's get started. Start by breeding adult zebrafish three nights prior to the planned experiment. To stage embryos at two days post fertilization, prepare E three medium using five millimolar sodium chloride, 0.17 millimolar potassium chloride, 0.33 millimolar calcium chloride, 0.33 millimolar magnesium sulfate, collect embryos and place them in a Petri dish with warmed E three water in an incubator at 28.5 degrees Celsius.

Remove any dead embryos from the Petri dish at the end of the first day. Separate embryos to 80 to 100 embryos per Petri dish. Prepare glass injection needles by pulling capillary tubes to a final diameter of approximately 10 to 20 microns with an electrode puller.

After pulling, place the capillary under a dissecting microscope and use a ruler to mark approximately ten one millimeter lines along the needle with a permanent marker. Store the needles in a large Petri dish on clay ramps. Next, prepare the holding pipette by fire.

Polishing the tip of a thin walled bora silicate glass capillary. Hold the glass capillary using long tongs and heat the tip of the capillary by placing it vertically over the light blue inner cone of the flame for one to two seconds. Rolling the capillary to obtain an even melt.

Repeat this a few times, then examine the capillary under the on a stage micrometer. The capillary should be 0.4 to 0.5 millimeters in diameter. After preparing capillaries and holders, fill the manual micro syringe pump with mineral oil following the instructions given by the manufacturer.

Insert the pipette holder in a joystick manipulator connected with a magnetic stand to an iron plate. Prepare 20 microliters of the nephrotoxin injection mixture. 2.4 nanoliters of Gentamicin and 17.6 nanoliter of Lucifer yellow dextran in the control rva.

The gentamicin is replaced with saline solution. Prepare two 30 millimeter Petri dishes with two milliliters of mineral oil near the injection microscope to drop the nephrotoxic and control mixtures prior to injection. Prepare the anesthetic by dissolving 400 milligrams of trica powder in 97.7 milliliters of double distilled water and 2.1 milliliters of one molar triss and adjusting to pH seven.

Store this stock solution in the freezer in Eloqua of 4.2 milliliters. To use trica as an anesthetic, dilute the 4.2 milliliter trica stock solution in 100 milliliters of E three and leave it room temperature next to prepare larvae for injection under a dissecting microscope, manually remove the coons with fine forceps. Anesthetize the zebrafish larvae by transferring them to a Petri dish with trica warmed at room temperature.

Zebrafish larvae should be exposed to trica for at least 15 minutes. Before starting the micro injections, open the tip of the injection needle using a razor blade. Hold the blade at a slight angle to create a bevel and cut the tip such as to obtain a tip opening of about 10 to 20 microns in diameter.

Turn on the power and air supply for the microinjection apparatus. Insert the needle into the needle holder. Fill the needle by placing a 10 microliter droplet of the injection solution in the oil-filled Petri dish.

Under the microscope, turn on the manual pico pump and sub burge the tip of the needle. In the droplet, set the pico pump to vent to fill the needle. This will take a few minutes.

If the solution moves into the needle too fast, the diameter of the tip is too big and the injections won't be accurate. After the needle is filled, set the pico pump to hold to calibrate the needle, set the pico pump switch to gated. Press the foot pedal to discharge the needle and record the time it takes in seconds for the meniscus to travel from one millimeter marking to the next repeat three times and divide the average time by 30, which is the volume in nanoliters corresponding to the one millimeter linear distance and obtain the number of milliseconds required to deliver one nanoliter of solution.

Adjust the range period knob to this number and move the gated time switch to timed. Now the peal pump is set to deliver a one nanoliter pulse of solution. Next position the Petri dish with the larvae under the microscope.

Group the larvae in the center of the Petri dish and focus the microscope. Place the holding pipette in the manual micro syringe pump on the opposite side of the micro injector and advance its tip to the petri dish into the plane of focus of the microscope. Bend it with a fairly shallow angle so that the very tip of the holding pipette is touching the bottom of the Petri dish and is close to horizontal.

Change to a higher magnification on the microscope and use a super fine eyelash with handle. To position the larval zebra fish dorsal side down with the yolk very close to the holding pipette. Turn the micrometer drive controlling the manual micro syringe pump counterclockwise to hold the larval zebra fish by sucking the yolk into the holding pipette.

Take care not to turn the micrometer too far as the yolk can burst. If pulled too deep into the holding pipette, focus on the forming common cardinal vein, which lies over the yolk just beneath the periderm, and is a site of high blood flow. With the micro manipulator of the injection apparatus.

Guide the needle into the developing common cardinal vein. It is typically necessary to pull the needle back a bit after insertion as the common cardinal vein is very superficial. If the yoke of the larvae is properly held with the holding pipette, it won't roll as the needle enters.

Inject one nanoliter of solution by pressing the foot pedal. Then release the larvae from the holding pipette and return it to the larvae water. To verify the success of the microinjection, monitor the presence of the Lucifer yellow dextre in the heart of the zebrafish larvae.

Under a fluorescent microscope, the Lucifer yellow remains visible in the circulatory system for approximately 20 minutes, return the larvae to fresh E three medium at four days post fertilization. Put between 10 and 15 larval zebra fish in four milliliter glass vials and fix them in one milliliter of dents for four hours at room temperature. Rehydrate the larvae in a graded methanol P-B-T-P-B-S with 0.5%between 20 series 75 to 25, 50 to 50 25 to 75 100%PBT pipetting the solution out and adding new solution to the same vial.

One milliliter of solution per vial. 20 minutes in each solution. Larvae should never be exposed to air.

Always leave a little solution covering the larvae. When changing solutions, remove PBT and replace with one milliliter of blocking solution PBS with 1%DMSO 0.5%between 20 10%normal goat serum for three hours at four degrees Celsius on a mutator. Transfer the larvae in a two milliliter eph.

Remove blocking serum and replace with 300 microliters of primary antibody. A monoclonal sodium potassium at TPS at one to 25 dilution alpha six F supernat, and 300 microliters of incubation solution PBS with 1%DMSO 0.5%tween 22%normal goat serum overnight at four degrees Celsius on a mutator. Remove the primary antibody and wash three times for 30 minutes each with one milliliter of incubation solution at room temperature placing on a mutator for each wash.

Then incubate with secondary antibody diluted one to 500 in incubation solution for two hours at room temperature After this wash, three times for 30 minutes each with PBT, dehydrate the larvae through a graded ethanol series soaking for 10 minutes per step. Prepare the infiltration solution by using 25 milliliters of JB four embedding solution A and 0.24 grams of catalyst dissolving for 20 to 30 minutes. The solution can be stored at four degrees Celsius up to one month.

Remove the 100%ethanol and replace with plastic infiltration solution. Place the tubes at four degrees Celsius on a rotator and allow infiltration to proceed for about 30 minutes until the larvae sink to the bottom of the tube. To embed larvae, place the plastic molding cup tray under a dissecting microscope.

Transfer the larvae along with enough infiltration solution to cover the larvae into the cups. The plastic embedding media is prepared in a small plastic tube. We normally embed four to five larvae at a time using one larvae for each cup and prepare five milliliters of embedding plastic.

Add 35 microliters of JB four, embedding solution B For each milliliter of infiltration solution mix well pipetting up and down with a plastic paste or pipette several times to ensure JB four will polymerize properly. After five milliliters of embedding plastic has been prepared, remove infiltration plastic from round samples with a paste pipette. Fill the cup with embedding plastic, the plastic must ooze out of the cup to ensure good attachment of the block holder to the specimen.

To detect zebra fish renal tubules transverse sections are the best for this purpose. The larvae must be embedded vertically. Head down with the aid of an eyelash with handle or fine forceps.

Periodically position the larvae vertically until plastic is hard enough that they don't move from their position. It takes about 20 to 30 minutes. Then place the EVH two block holder over the cups and leave the tray at four degrees Celsius overnight until polymerization is complete.

Set the specimen into the chuck holder of the microtome and set the slide warmer to 45 degrees Celsius. Pour deionized water into a beaker and place it close to the microtome before sectioning. Be sure the block is oriented such as to obtain perfect transverse sections of the larvae.

The orientation head adjustment levers can be rotated and the backlight age in positioning of the specimen. Cut thick sections through the head. Orient the blocks so that equivalent sections of the eyes are taken for each larva.

When the pectoral fins appear in the sections, that is when the prone tubules start. From this point, cut about 32 4 micron section slices from the blocks. Collect the sections with forceps and let them drop into the beaker with water without touching the water with the forceps.

The section will expand as soon as it hits the surface of the water. Now you can collect it on a slide. Insert the slide into the water at a 45 degree angle and approach the section with the help of an eyelash with handle touching only the borders of the section, incubate the slides on a slide warmer until completely dry, and image sections using epi fluorescent or confocal microscopy.

When performed correctly, an injected larva appears as shown here in the top image with the fluorescent luciferase yellow dextran in the vascular system of the larvae. If the injection is too deep, as in the lower image injected material can be seen in the yolk sac or the heart can be punctured causing excessive bleeding. The larvae developed pericardial edema as a consequence of gentamicin nephrotoxicity as seen here.

The control larvae at the top display no pericardial edema. However, when injected with gentamycin, 80%of the zebrafish larvae display moderate or severe edema as indicated by the arrows in the middle and bottom panels. We use a phenotype based classification system in which larvae with moderate edema show evident pericardial edema, but no other observable phenotypes.

Whereas severe edematous larvae show pericardial and trunk edema, lack of trunk circulation, and slight to moderate access curvature using a renal tubule antibody against sodium potassium atpa ace. The damaged tubules are visualized in transverse sections with an evident loss of cell polarity in panel C and D and tubular disruption in damaged tubules as compared to controls in panels A and B.This pattern is consistent through multiple transverse sections when comparing sections. Factorial fins and proximal convoluted tubules are used as anatomical markers.

We have just shown you how to induce acute kidney injury in larval zebrafish. When doing this procedure is important to remember to use injection needles with a diameter of 10 20 micrometers. It might be necessary to fire polish a few holding pipet in order to obtain the appropriate sides, but once you have one, you can just keep it because it can last for several injection sections.

Also, it's important to make sure not to have any air bubble in the micro series JA apparatus because this would decrease the position of the system. Finally, position the fish vertically during the embedding in order to obtain a transverse perfect transverse section of the renal tubal. So that's it.

Thanks for watching and good luck with your experiments.