The overall goal of this procedure is to induce targeted ablation of Zebrafish kidney epithelia cells to model nephron injury and repair. This method can help answer key questions in the organ and specifically kidney regeneration field. Such as, what are the major cellular processes involved in regeneration, what is the extent of plasticity of surviving tissues and many more.
The main advantage of this technique is that it allows for precise spatial temporal control of injury, as well as the ability to adjust the level of injury. The implications of this technique extend toward therapy or diagnosis of acute kidney injury, because it allows precise spatial and temporal assessment of cellular and molecular processes involved in kidney cell injury and regeneration. To begin, generate embryos by crossing kidney GFP fluorescent Zebrafish.
For 24 hours following fertilization, keep the embryos in E3 solution at 28.5 degree celsius. Then use 0.003%PTU in E3, to replace the E3 medium. Return the embryos to the incubator for six or more days, at which point the kidneys would have matured.
To map the Zebrafish for live imaging, prepare 1%to 2%low melting agarose with 0.2 milligrams per milliliter trigaine solution. Ensure the agarose trigaine solution is cool before using a plastic or glass pipette to transfer an embryo into the solution. Next, using a transfer pipette, draw up the larva in the agarose and deposit it in the middle of a 35 millimeter dish.
Quickly spread the agarose containing the Zebrafish to evenly cover the bottom of the dish. Then use a glass probe to orient the larva for photo ablation and imaging, so that the kidney segment of interest is perpendicular to the beam path, while the contra lateral segment is away from the laser beam. Orientation of the Zebrafish is crucial to the proceeding steps involving laser ablation.
A good technique is advise to ensure that the larvae maintains a perpendicular position in the agarose. Cover the Petri dish to minimize evaporation and allow the agarose to solidify for about 15 minutes. After setting up the confocal microscope, according to the text protocol, in the upright configuration, position the Petri dish containing the larva on top of a microscope slide and use modeling clay to keep it in place.
Next, position the sliding Petri dish on the microscope stage. Then, add three milliliters of imaging solution. Under a water dipping lens, slowly raise the stage, making sure the lens is slightly angled to prevent air from becoming trapped in the beam path.
Once the lens touches the solution at an angle, center it so that it is in the field of view at the embryo. Now, using the fluorescent light source, find the segment of interest, then, focus on the superficial branch to target it for injury. The superficial branches chosen to minimize light scattering.
In the microscope software, navigate to view, acquisition control, C2plus compact GUI. Set the pixel dwell time to 1.9 microseconds, the frame size to 512 by 512 pixels and the pinhole size to 90 microns. Set the 405 nanometer laser intensity to zero and the 488 laser intensity to a low value.
While using the 488 nanometer laser, click scan in the software interface. Find the segment of interest that is perpendicular to the beam path and that is well visualized with good GFP fluorescence. Once that region has been identified, navigate to ROI, draw elliptic ROI, to draw a region of interest.
Next, navigate to view, acquisition controls, C2plus scan area and choose band scan area, the rectangular mask will appear within that interface. Using the cursor, manually define a rectangular scan to cover the segment targeted for laser ablation. Right click within the mask area, to accept the selection.
Using just the 488 nanometer laser to activate GFP, begin the time measurement while monitoring the green channel. Ensure that the intensity of the 488 laser is relatively low. Measure the average intensity of the GFP in the region of interest by selecting measure, time measurement.
Use the graph or data tab to monitor the average intensity levels within the ROI. Using the violet laser, induce damage to the kidney cells by increasing the intensity to 100%by sliding the laser power control, all the way to the right. Within the graph tab, observe a line graph or use the data tab to observe the numerical values of GFP intensity and wait until it drops to a desired level.
For example, 50%of base line. Once the GFP intensity within the elliptical ROI has dropped to a desired level, immediately decrease the intensity of the violet laser to zero in the software control panel by moving the laser power slider, all the way to the left. Finally, obtain a new baseline of GFP fluorescence.
As shown in this figure, after exposure to 405 nanometer laser light, GFP fluorescence continues to disappear one cell at a time. Until at 220 minutes, the entire ablate segment loses 100%of its GFP positivity. This is due to cell death and not the down regulation of GFP expression, as seen by the appearance of red fluorescent propidium iodide positive nuclei in the cells that lose GFP positivity.
These images demonstrate that by varying the initial exposure, it is possible to dial the injury and the response to the injured epithelium. With 10%to 20%initial GFP photo bleaching, virtually no reduction in GFP positivity is observed at five hours post injury. 50%and 60%leads to a complete disappearance of GFP at five hours, while 30%and 40%bleaching leads to intermediate results with 50%estimated death observed at about 35%photo bleaching.
This is supported by propidium iodide staining where 20%photo bleaching results in virtually no propidium iodide staining at 100 minutes post ablation. However, 50%photo bleaching leads to continuous propidium iodide positivity in the ablated epithelium, after 60 minutes. While 30%and 40%photo bleaching leads to intermediate propidium iodide incorporation.
Once mastered, this technique can be done in 30 minutes if it is performed properly. Don't forget that working with lasers and chemical reagents can be extremely hazardous and pre-cautions such as, avoiding looking at the laser beam and using gloves when handling chemicals should always be taken while performing this procedure. Following this procedure, other methods like time lapse microscopy, immunofluorescence staining and others can be performed in order to answer additional questions aimed at dissecting kidney injury and repair at the cellular and molecular level.
After its development, this technique paved the way for researchers in the field of kidney regeneration. To explore the role of collective cell migration and kidney repair after injury in Zebrafish. After watching this video, you should have a good understanding of how to use confocal microscopy to injure specific kidney segments in GPF transgenic Zebrafish larvae and embryos.
