The overall goal of this procedure is to perform targeted celebration of kidney cells in the zebrafish embryonic kidney, the pro nephros for the purpose of studying renal epithelial regeneration following acute kidney injury. This is accomplished by first micro injecting zebrafish embryos with a dextran fluorescent conjugate. During an overnight incubation, the conjugates are selectively absorbed by nephron proximal tubial cells.
The next day, the cells are examined under epi fluorescence and selected cells are ablated using a pulsed laser system. Later, morphological and molecular changes are assessed using histology, whole mounting C two hybridization and immunohistochemistry chemistry. The main advantage of this technique over existing methods like acute kidney injury using the nephrotoxic antibiotic gentamicin, is that such treatments trigger catastrophic damage and are lethal precluding the analysis of renal regeneration events over time.
This method can help answer key questions in the kidney field, such as the identification of molecular signaling events that occur during epithelial regeneration. We first had the idea for this technique when we wanted to develop a detailed analysis of renal epithelial regeneration In Petri dishes. Collect fertilized zebrafish embryos in modified E three solution without methylene blue.
Raise the fertilized embryos at 28.5 degrees Celsius until they're between 48 and 55 hours Post fertilization when the embryo's renal system has begun to function before beginning the injections, prepare an aros injection tree using a mold with triangular depressions to hold the embryos. Next, prepare standard microinjection needles and an injection solution to label the proximal tubule. Begin the experiment by preparing the embryos.
First, remove the Corian from any unhatched eggs using fine forceps. Then rinse the Corian debris, debris from the Petri dish and refill the dish with 30 milliliters of modified E three solution. Second, anesthetize the larvae with five milliliters of trica.
Make sure the larvae are completely anesthetized by checking for a touch response. If they are not fully anesthetized, they would twitch during the injection. Third, using a plastic transfer pipette, put the larvae in the mold.
Position the animals with their heads in the deepest part of the depression and their trunks along the side of the well. Now proceed with intramuscular micro injections by loading a prepared needle with two to three microliters of dextran fluoresce in position the needle and inject approximately one nanoliter of solution into a trunk so mite of each animal. The dextran conjugates are readily endocytosed by the nephron proximal tubule enabling preferential labeling of this epithelial cell population.
Aim for the upper portion of the so mite and avoid the yolk sac extension, or the nephron tubules will end up disrupted, such as in this improperly injected animal. Once the larvae are injected, transfer them to a clean Petri dish and rinse them three times with fresh E three media to remove all traces of trica. Now, cover the lid with foil and incubate the animals overnight at 28 degrees Celsius.
Before beginning this procedure, prepare Im mobilization media and to keep some aliquots of it for immediate use at room temperature and to keep the remaining aliquots at four degrees Celsius for long-term storage. Begin by anesthetizing the zebra fish larvae with five milliliters of trica in 30 milliliters of E three. Just as before, make certain the fish do not respond to tactile sensation.
Now examine the injected animals under fluorescence to select larvae with easily visualized proximal tubules. Transfer up to 12 larvae to a new dish containing the same concentration of trica at a compound fluorescent microscope outfitted with a fitzy filter, brightfield lighting, and a laser perform. The ablation begin by transferring and anesthetize lava to a dish containing immobilization media.
And wait two minutes, then transfer the lava to a glass depression slide containing a drop of immobilization media. Gently position the animal dorsal side up with a fine probe and focus on the dorsal side. Now using a calibrated pulsed micro point laser system abl one of the nephrons leaving the other intact as a control.
You'll see dispersal of the fluorescence as the renal epithelial cells are ablated. Then gently transfer the animal to a well of a 12 world dish and rinse it three times in E three media to wash away the methyl cellulose. Continue ablating nephrons until the first 12 world dish is filled.
Then anesthetize another 12 animals and repeat the process. Dextra Fitzy was injected to preferentially label the proximal tubule. Three days post fertilization and nephron of each animal was laser ablated.
One day after ablation. Regeneration is not apparent four days after ablation, however regeneration has occurred following cell ablation in situ two. Hybridization was used to analyze changes in fixed samples at single time points.
This embryo was not subjected to ablation. In contrast, this embryo had a large stretch of tubial epithelium ablated, and this embryo had a short interval of tubular epithelium ablated. Following this procedure, other methods like hol mount situ hybridization can be performed in order to answer additional questions like the identity of gene expression changes during the course of nephron regeneration.
Don't forget that working with chemicals can be extremely hazardous. Precautions, such as wearing personal protective equipment like a lab coat and gloves are recommended during this procedure.
