We would like to thank Dr. Iain Drummond and Dr. Vladimir Korzh for sharing kidney GFP transgenic lines. We would also like to thank NYITCOM for providing necessary resources to conduct this work. This study was in part supported by grants: K08DK082782, R03DK097443 (NIH), and the HSCI Pilot Grant (AV).

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the NYIT College of Osteopathic Medicine Institutional Animal Care and Use Committee (NYITCOM IACUC). All surgery and in vivo experimentation was performed under tricaine anesthesia, and all efforts were made to minimize suffering.
1. Obtaining and Maintaining Embryos
<ol>
	<li>Obtain embryos by c...

<ol>
	<li>Chertow, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Kidney+Injury,+Mortality,+Length+of+Stay,+And+Costs+In+Hospitalized+Patients.">Acute Kidney Injury, Mortality, Length of Stay, And Costs In Hospitalized Patients.</a> J Am Soc Nephrol. 16 (11), 3365-3370 (2005).</li><li>Yasuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+And+Clinical+Outcomes+of+Acute+Kidney+Injury+Requiring+Renal+Replacement+Therapy+In+Japan.">Incidence And Clinical Outcomes of Acute Kidney Injury Requiring Renal Replacement Therapy In Japan.</a> Ther Apher Dial. 14 (6), 541-546 (2010).</li><li>Waikar, S. S., Liu, K. D., Chertow, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+Epidemiology+And+Outcomes+of+Acute+Kidney+Injury.">Diagnosis, Epidemiology And Outcomes of Acute Kidney Injury.</a> Clin J Am Soc Nephrol. 3 (3), 844-861 (2008).</li><li>Lameire, N., Van Biesen, W., Vanholder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Kidney+Injury.">Acute Kidney Injury.</a> The Lancet. 372 (9653), 1863-1865 (2008).</li><li>Esson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+And+Treatment+of+Acute+Tubular+Necrosis.">Diagnosis And Treatment of Acute Tubular Necrosis.</a> Ann Intern Med. 137 (9), 744 (2002).</li><li>Vaidya, V. S., Ferguson, M. A., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+Acute+Kidney+Injury.">Biomarkers of Acute Kidney Injury.</a> Annu. Rev. Pharmacol. Toxicol. 48 (1), 463-493 (2008).</li><li>Hentschel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Renal+Failure+In+Zebrafish:+A+Novel+System+To+Study+A+Complex+Disease.">Acute Renal Failure In Zebrafish: A Novel System To Study A Complex Disease.</a> Am J Physiol Renal Physiol. 288 (5), F923-F929 (2005).</li><li>Cianciolo Cosentino, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+Microinjections+of+Zebrafish+Larvae+To+Study+Acute+Kidney+Injury.">Intravenous Microinjections of Zebrafish Larvae To Study Acute Kidney Injury.</a> J Vis Exp. (42), (2010).</li><li>Johnson, C. S., Holzemer, N. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Ablation+of+The+Zebrafish+Pronephros+To+Study+Renal+Epithelial+Regeneration.">Laser Ablation of The Zebrafish Pronephros To Study Renal Epithelial Regeneration.</a> J Vis Exp. (54), (2011).</li><li>Drummond, I. A., Davidson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Kidney+Development.">Zebrafish Kidney Development.</a> Methods Cell Biol. , 233-260 (2010).</li><li>Toback, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+After+Acute+Tubular+Necrosis.">Regeneration After Acute Tubular Necrosis.</a> Kidney Int. 41 (1), 226-246 (1992).</li><li>Witzgall, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Proliferating+Cell+Nuclear+Antigen,+Vimentin,+C-Fos,+And+Clusterin+In+The+Postischemic+Kidney.+Evidence+For+A+Heterogenous+Genetic+Response+Among+Nephron+Segments,+And+A+Large+Pool+of+Mitotically+Active+And+Dedifferentiated+Cells.">Localization of Proliferating Cell Nuclear Antigen, Vimentin, C-Fos, And Clusterin In The Postischemic Kidney. Evidence For A Heterogenous Genetic Response Among Nephron Segments, And A Large Pool of Mitotically Active And Dedifferentiated Cells.</a> J Clin Invest. 93 (5), 2175-2188 (1994).</li><li>Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiation+And+Proliferation+of+Surviving+Epithelial+Cells+In+Acute+Renal+Failure.">Dedifferentiation And Proliferation of Surviving Epithelial Cells In Acute Renal Failure.</a> J Am Soc Nephrol. 14 (90001), (2003).</li><li>Palmyre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+Epithelial+Migration+Drives+Kidney+Repair+After+Acute+Injury.">Collective Epithelial Migration Drives Kidney Repair After Acute Injury.</a> PLoS ONE. 9 (7), e101304 (2014).</li><li>Vasilyev, A., Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+Kidney+Development+In+Zebrafish.">Live Imaging Kidney Development In Zebrafish.</a> Methods Mol Biol. , 55-70 (2012).</li><li>Korzh, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Compound+Transgenic+Zebrafish+In+Development:+A+Tale+of+Green+Fluorescent+Protein+And+Killerred+.">Visualizing Compound Transgenic Zebrafish In Development: A Tale of Green Fluorescent Protein And Killerred .</a> Zebrafish. 8 (1), 23-29 (2011).</li><li>Bollig, F., Mehringer, R., Perner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+comparative+expression+analysis+of+a+second+wt1+gene+in+zebrafish.">Identification and comparative expression analysis of a second wt1 gene in zebrafish.</a> Dev Dyn. 235 (2), 554-561 (2006).</li><li>Liu, Y., Pathak, N., Kramer-Zucker, A., Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+controls+the+differentiation+of+transporting+epithelia+and+multiciliated+cells+in+the+zebrafish+pronephros.">Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros.</a> Development. 134 (6), 1111-1122 (2007).</li><li>Diep, C. Q., Ma, D., Deo, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+adult+nephron+progenitors+capable+of+kidney+regeneration+in+zebrafish.">Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.</a> Nature. 470 (7332), 95-100 (2011).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebra+fish.">Characterization of mesonephric development and regeneration using transgenic zebra fish.</a> Am J Physiol. 299 (5), F1040-F1047 (2010).</li><li>Fisher, S., Grice, E. A., Vinton, R. M., Bessling, S. L., McCallion, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+RET+regulatory+function+from+human+to+zebrafish+without+sequence+similarity.">Conservation of RET regulatory function from human to zebrafish without sequence similarity.</a> Science. 312 (5771), 276-279 (2006).</li><li>Seiler, C., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+labeling+of+the+zebrafish+pronephric+duct+and+tubules+using+a+promoter+from+the+enpep+gene.">Transgenic labeling of the zebrafish pronephric duct and tubules using a promoter from the enpep gene.</a> Gene Expr Patterns. 11, 118-121 (2011).</li><li>Lin, H. F., Traver, D., Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thrombocyte+development+in+CD41-GFP+transgenic+zebrafish.">Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.</a> Blood. 106 (12), 3803-3810 (2005).</li><li>Choo, B. G., Kondrichin, I., Parinov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+transgenic+Enhancer+TRAP+line+database+(ZETRAP).">Zebrafish transgenic Enhancer TRAP line database (ZETRAP).</a> BMC Dev Biol. 6 (1), 5 (2006).</li><li>Parinov, S., Kondrichin, I., Korzh, V., Emelyanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tol2+transposon-mediated+enhancer+trap+to+identify+developmentally+regulated+zebrafish+genes+in+vivo.">Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.</a> Dev Dyn. 231 (2), 449-459 (2004).</li></ol>

The authors declare that they have no competing financial interests.

It should be noted that the total laser power varies between systems. However, using percent GFP photobleaching allows for a readout of total energy delivered to the fluorescent kidney, independent of the variation in laser power and compensated for by the length of exposure. Keep in mind, however, that the response of different tissues to this method of photoablation varies. Even during kidney maturation, it is significantly more difficult to obtain a 50% reduction in GFP fluorescence in younger embryos than in mature l...

Acute Kidney Injury (AKI)1,2, which also can be referred to as acute renal failure, is broadly defined as a sudden impairment in kidney function3. While the level of understanding of this condition has been enhanced remarkably over the years, morbidity and mortality rates have remained high1,2. The current treatment for this condition is mostly supportive, as results from multiple clinical trials of drug therapy have been negative4,5. The kidney is unique in that it has the ability to repair itself. Therefore, supportive therapy after an early diagnosis of AKI is the best way to limit morbidity6. However, it is difficult to detect AKI early, and the mortality rate is a staggering 50-80% for those who require dialysis5. With the ability of the kidneys to repair themselves and the lack of treatment options for this condition, it is important to develop methods to enhance this nephron regeneration process.
There have been many different models used for AKI research that includes different agents of injury and animal models. In terms of agents of kidney damage, the aminoglycoside antibiotic gentamicin has been used as a nephrotoxic agent that leads to AKI7,8. However, several groups have found that gentamicin treatment is lethal to the zebrafish embryo9. It causes tubular damage that is too serious for embryo recovery, making the study of regeneration difficult without some type of intervention. Mammalian models, like the mouse and rat, are also considered valuable, but they face many limitations during the study of AKI. Perhaps the main disadvantage of rodent models is the difficulty in visualizing the rodent kidney and thus determining the precise spatiotemporal processes leading to epithelial death and repair.
Johnson et al. have reported a laser ablation-based technique to induce acute kidney injury in embryonic and larval zebrafish9. They used pulsed laser ablation to damage the kidney after an intramuscular injection with dextran conjugates. The fluorescence from dextran conjugates allows for the visualization of damage and regeneration in the tubule epithelium9. This model overcomes the two limitations mentioned above, but it does not allow for graded levels of injury and is difficult to carry out on large, arbitrary cell groups.
The new laser ablation-based zebrafish model of AKI described here addresses all of the above limitations. The pronephric kidney in larval zebrafish is a mature, functioning organ that contains segments similar to the mammalian nephron, including a glomerulus, proximal and distal tubules, and a collecting duct10. Zebrafish larvae are also optically transparent, making it feasible to observe the kidney through fluorescence techniques. Thus, zebrafish are a valuable in vivo model of AKI, and the larval pronephric kidney (5-12 days-post-fertilization (dpf)) can be used to study the cellular and molecular processes involved in kidney injury and repair.
This paper presents a method through which specific Green Fluorescent Protein (GFP)-expressing nephron segments can be photoablated using a low-energy (compared to a pulsed-laser system) violet laser light (405 nm). The GFP fluorescence allows for the targeting of a group of cells, making the changes that occur visible through the observation of GFP photobleaching. In addition, GFP (by absorbing violet light) serves as an energy sink to potentiate the injury in GFP-expressing kidney cells. Time-lapse microscopy can then be used to study the repair process. Studies have found cell proliferation, cell migration, and cell metaplasia11,12,13 to all be potential processes that may play an important role in kidney repair. However, the relative importance of these processes and the details of their interplay have been difficult to uncover due to the limitations of existing models of AKI. Using this novel approach, it was possible to show that cell migration plays a central role in kidney repair after acute injury14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Petri Dishes, 35 x 10mm</td>
			<td>Genesee Scientific</td>
			<td>32-103</td>
			<td>Procedural Usage: Step 2.4,2.7</td>
		</tr>
		<tr>
			<td>Petri Dishes, 100 x 15mm</td>
			<td>Midwest Scientific</td>
			<td>910</td>
			<td>Procedural Usage: Step 1</td>
		</tr>
		<tr>
			<td>De-chorination forceps- Electron Microscopy Sciences Dumont Tweezers 5 Dumostar</td>
			<td>Fischer Scientific</td>
			<td>50-241-57</td>
			<td>Procedural Usage: Step 2.1.1</td>
		</tr>
		<tr>
			<td>Plastic Transfer Pipet</td>
			<td>Globe Scientific</td>
			<td>135030</td>
			<td>Procedural Usage: Step 2.5, 3.6</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma Aldrich</td>
			<td>A5040-25G</td>
			<td>Procedural Usage: Step 2.3, 3.4</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fischer Scientific</td>
			<td>BP165-25</td>
			<td>Procedural Usage: Step 2.3</td>
		</tr>
		<tr>
			<td>Pulled glass probe (manufactured manually from glass capillary tubes)</td>
			<td>Fischer Scientific</td>
			<td>21-1640-2C</td>
			<td>Procedural Usage: Step 2.4</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1270</td>
			<td>Procedural Usage: Step 1.5</td>
		</tr>
		<tr>
			<td>SOLA Light Engine</td>
			<td>Lumencor</td>
			<td>SOLA SM-5-LCR-SB</td>
			<td>Procedural Usage: Step 1.5</td>
		</tr>
		<tr>
			<td>Eclipse C2 Plus Confocal Microscope System</td>
			<td>Nikon</td>
			<td></td>
			<td>Procedural Usage: Step 3</td>
		</tr>
		<tr>
			<td>1x E3 Solution</td>
			<td></td>
			<td></td>
			<td>Recipe used to generate: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 Procedural Step Usage: 1.2, 1.3, 2.2, 2.3</td>
		</tr>
		<tr>
			<td>PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td>Procedural Step Usage: 1.3, 2.2, 3.4, and 4.2</td>
		</tr>
		<tr>
			<td>NIS Elements Software</td>
			<td>Nikon</td>
			<td>C2+</td>
			<td>Procedural Usage: Step 3</td>
		</tr>
		<tr>
			<td>Laser Unit</td>
			<td>Agilent</td>
			<td>MLC 400</td>
			<td>Procedural Step 3.11</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4170-100MG</td>
			<td>Procedural Step Usage: 4.2</td>
		</tr>
	</tbody>
</table>

precise cellular ablation approach for modeling acute kidney injury in developing zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

Please note that this protocol was successfully used with a number of kidney GFP transgenic lines, including ET(krt8:EGFP)sqet11-9, ET(krt8:EGFP)sqet33-d10, and Tg(atp1a1a.4:GFP). The example results shown here were obtained using the ET(krt8:EGFP)sqet11-9 line.
Figure 1 shows the example photoablation protocol. The average GFP intensity is monitored inside the region of interest (<strong class="xfi...

Watch this Scientific Journal Video about Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish at JoVE.com

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

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.

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6.6K ビュー.  NYITCOM. 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 a...