Name: in situ hybridization in zebrafish larvae and juveniles during mesonephros development
Uploaded: 2021-08-13T14:45:00
Description: Watch this Scientific Journal Video about In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development at JoVE.com

Funding was provided by the Pennsylvania Academy of Science, and the Commonwealth of Pennsylvania Biologists, and the Indiana University of Pennsylvania (School of Graduate Studies and Research, Department of Biology, and the Cynthia Sushak Undergraduate Biology Fund for Excellence). The Tg(lhx1a-EGFP) transgenic line was provided by Dr. Neil Hukriede (University of Pittsburgh).

The use of zebrafish larvae and juveniles is approved by the IUP IACUC (protocol #02-1920, #08-1920). Details of the solution content are listed in the Table of Materials.
1. Raising larvae
NOTE: It will take up to 21 days or more to raise larvae and juveniles to the stage of interest.
<ol>
	<li>Set up adult zebrafish to mate by adding 1 male and 1 female fish in a mating tank in the late afternoon after their last meal. 
	&#8203;NOTE: Not al...

<ol>
	<li>Kinth, P., Mahesh, G., Panwar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+zebrafish+research:+a+global+outlook.">Mapping of zebrafish research: a global outlook.</a> Zebrafish. 10 (4), 510-517 (2013).</li><li>Grunwald, D. J., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Headwaters+of+the+zebrafish+-+emergence+of+a+new+model+vertebrate.">Headwaters of the zebrafish - emergence of a new model vertebrate.</a> Nature Reviews. Genetics. 3 (9), 717-724 (2002).</li><li>Wingert, R. 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=The+zebrafish+pronephros:+a+model+to+study+nephron+segmentation.">The zebrafish pronephros: a model to study nephron segmentation.</a> Kidney International. 73 (10), 1120-1127 (2008).</li><li>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=Kidney+development+and+disease+in+the+zebrafish.">Kidney development and disease in the zebrafish.</a> Journal of the American Society of Nephrology: JASN. 16 (2), 299-304 (2005).</li><li>Swanhart, L. M., 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+kidney+development:+basic+science+to+translational+research.">Zebrafish kidney development: basic science to translational research.</a> Birth defects research. Part C, Embryo Today: Reviews. 93 (2), 141-156 (2011).</li><li>Dressler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</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> Journal of Visualized Experiments: JoVE. (54), e2845 (2011).</li><li>Marra, A. N., 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+gene+expression+during+zebrafish+pronephros+development+and+regeneration.">Visualizing gene expression during zebrafish pronephros development and regeneration.</a> Methods in Cell Biology. 154, 183-215 (2019).</li><li>McMenamin, S. K., Chandless, M. N., Parichy, 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=Working+with+zebrafish+at+postembryonic+stages.">Working with zebrafish at postembryonic stages.</a> Methods in Cell Biology. 134, 587-607 (2016).</li><li>Diep, C. Q., 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=Development+of+the+zebrafish+mesonephros.">Development of the zebrafish mesonephros.</a> Genesis. 53 (3-4), 257-269 (2015).</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+zebrafish.">Characterization of mesonephric development and regeneration using transgenic zebrafish.</a> American Journal of Physiology. Renal Physiology. 299 (5), 1040-1047 (2010).</li><li>Diep, C. Q., 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>Diep, C. Q., Mikeasky, N., Davidson, A. J., Cartner, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish in Biomedical Research: Biology, Husbandry, Diseases, and Research Applications. , 145-150 (2020).</li><li>Parichy, D., Elizondo, M., Mills, M., Gordon, T., Engeszer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Developmental dynamics: An Official Publication of the American Association of Anatomists. 238 (12), 2975-3015 (2009).</li><li>Guo, 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=LIM+Homeobox+4+(lhx4)+regulates+retinal+neural+differentiation+and+visual+function+in+zebrafish.">LIM Homeobox 4 (lhx4) regulates retinal neural differentiation and visual function in zebrafish.</a> Science Reports. 11 (1), 1977 (2021).</li><li>Vauti, F., Stegemann, L. A., Vogele, V., Koster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-age+whole+mount+in+situ+hybridization+to+reveal+larval+and+juvenile+expression+patterns+in+zebrafish.">All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish.</a> PloS One. 15 (8), 0237167 (2020).</li><li>Parichy, D. M., Turner, J. M., Parker, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+puma+in+development+of+postembryonic+neural+crest-derived+cell+lineages+in+zebrafish.">Essential role for puma in development of postembryonic neural crest-derived cell lineages in zebrafish.</a> Developmental Biology. 256 (2), 221-241 (2003).</li><li>Yang, H., Wanner, I. B., Roper, S. D., Chaudhari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+in+situ+hybridization+with+signal+amplification+that+allows+the+detection+of+rare+mRNAs.">An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs.</a> The Journal of Histochemistry &amp; Cytochemistry. 47 (4), 431-445 (1999).</li><li>McCampbell, K. K., Springer, K. N., 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=Analysis+of+nephron+composition+and+function+in+the+adult+zebrafish+kidney.">Analysis of nephron composition and function in the adult zebrafish kidney.</a> Journal of Visualized Experiments: JoVE. (90), e51644 (2014).</li></ol>

The in situ hybridization method described here is aimed toward studying mesonephros development. However, it can be applied to study the development of other tissues and organs during metamorphosis, such as the gut, nervous system, scales, and pigmentation14. Probes can be generated for endogenous genes or fluorescent markers in transgenic lines.
It is critical for the larvae to remain intact in order to observe the organs and tissues in their native context. ...

The zebrafish embryo is an important model for studying tissue development due to its small size, transparency, available tools, and survival without feeding for up to five days1,2. It has greatly contributed to the understanding of kidney development and the conservation between zebrafish and mammals3,4,5. The kidney plays an essential role in maintaining fluid homeostasis, filtering the blood, and excreting waste6. The nephron, the functional unit of the kidney, comprises a blood filter connected to a long tubule. In zebrafish, two kidney structures form throughout its life. The pronephros (the temporary embryonic kidney) forms first during early development. It consists of two nephrons running along the anterior-posterior axis and becomes functional at around 2 days post fertilization (dpf). The utility of the pronephros lies in its simplicity, having just two nephrons that are mostly linear and easy to visualize (although the proximal convoluted tubule begins to coil at three dpf)3. This has facilitated not only studies of its early development from the intermediate mesoderm, but also the segmentation pattern and tubule repair7,8.
The usefulness of zebrafish becomes limited after five dpf, when the yolk is diminished and the larvae rely on feeding in the aquatic system9. At around 2 weeks old, the larvae undergo metamorphosis into juveniles, where new tissues form and old tissues are lost and/or reorganized9. This is also when the mesonephros (the permanent adult kidney) forms10,11,12. The first adult nephron forms near the sixth somite, and fuses with the distal early tubule of the pronephros. Additional nephrons are added posterior to this position initially, but also toward the anterior later on. The primary nephrons in this first wave fuse to the tubules of the pronephros and share a common lumen to deposit their waste. Secondary nephrons form in the next wave and fuse to primary nephrons. This reiterative process creates a mesonephros that is branched, somewhat akin to the mammalian kidney. Presumably, the pronephros eventually loses its tubule identity and becomes two major collecting ducts where all nephrons drain their waste13.
Prior to the formation of the first adult nephron, single progenitor cells begin to appear in ~4 mm (total length) larvae (~10 dpf). These cells, which are marked in the Tg(lhx1a-EGFP) transgenic line, adhere to the pronephros and seem to migrate to future sites of nephrogenesis. The single cells coalesce into clusters, which differentiate into new nephrons12. It is unclear where these cells reside or what genes they express before the onset of mesonephrogenesis.
Understanding mesonephros development provides insights into the mammalian kidney in ways that the pronephros cannot. These include the formation of nephrogenic aggregates from single progenitor cells, functional integration of new nephrons with existing ones, and branching morphogenesis. However, there are limitations to studying postembryonic development. The larvae are less transparent due to their larger size and having pigmentation. The developmental timeline is not synchronous among individual animals and is highly dependent on environmental factors, such as feeding and crowding9,14. Although knockdown reagents exist, they are less effective in larvae compared to embryos15. In this protocol, an in situ hybridization method to determine gene expression during zebrafish mesonephros development is described. Several steps are optimized to increase visualization of the mesonephros and penetration and washing of the probe and antibody. The Tg(lhx1a-EGFP) transgenic line is used along with a probe for EGFP to label single progenitor cells, nephrogenic aggregates, and the distal tubules of nascent nephrons. This method will provide a deeper understanding of mesonephros development and insight into regenerative therapies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-fluorescein antibody</td>
			<td>Roche/Sigma-Aldrich</td>
			<td>11426338910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>0.8% KOH, 0.9% H2O2 in PBST</td>
		</tr>
		<tr>
			<td>Blocking reagent</td>
			<td>Roche/Sigma-Aldrich</td>
			<td>11096176001</td>
			<td>Use for blocking solution, prepare according to manufacture&#39;s instruction</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Fisher Scientific</td>
			<td>&#160;22-363-549</td>
			<td>100 &#956;m</td>
		</tr>
		<tr>
			<td>E3 medium</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.17 mM KCl, 0.0001% methylene blue</td>
		</tr>
		<tr>
			<td>Eyelash manipulator</td>
			<td>Fisher Scientific</td>
			<td>NC1083208</td>
			<td>Use to manipulate larvae</td>
		</tr>
		<tr>
			<td>Fixing solution</td>
			<td></td>
			<td></td>
			<td>4% paraformaldehyde, 1% DMSO in PBS; heat at 65&#176;C while shaking until the powder dissolves, then add DMSO after it cools down</td>
		</tr>
		<tr>
			<td>Fluorescein probe synthesis</td>
			<td>Roche/Sigma-Aldrich</td>
			<td>11685619910</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>Fisher Scientific</td>
			<td>03-338B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hatchfry encapsulation</td>
			<td>Argent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hyb- solution</td>
			<td></td>
			<td></td>
			<td>50% formamide, 5X SSC, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Hyb+ solution</td>
			<td></td>
			<td></td>
			<td>HYB-, 5 mg/mL torula RNA, 50 ug/mL heparin</td>
		</tr>
		<tr>
			<td>MAB (10X)</td>
			<td></td>
			<td></td>
			<td>1 M maleic acid, 1.5 M NaCl, pH 7.5</td>
		</tr>
		<tr>
			<td>MABT</td>
			<td></td>
			<td></td>
			<td>1X MAB, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td></td>
			<td></td>
			<td>8% NaCl, 0.2% KCl, 1.44% Na2HPO4, 0.24% KH2PO4</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>PBST2</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.2% Tween-20</td>
		</tr>
		<tr>
			<td>Powder food</td>
			<td></td>
			<td></td>
			<td>Mix 2 g of each of spirulina and hatchfry encapsulon in 50 mL of fish system water and shake well</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P5568</td>
			<td>Use to permeabilize larvae</td>
		</tr>
		<tr>
			<td>Proteinase K solution</td>
			<td></td>
			<td></td>
			<td>20&#160; &#956;g/mL, 1% DMSO final concentration in PBST</td>
		</tr>
		<tr>
			<td>Spirulina microfine powder</td>
			<td>Argent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SSC (20X)</td>
			<td></td>
			<td></td>
			<td>3 M NaCl, 0.3 M sodium acetate anhydrous, pH 7, autoclave</td>
		</tr>
		<tr>
			<td>SSCT (0.2X)</td>
			<td></td>
			<td></td>
			<td>Dilute from 20X SSC, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>SSCT (2X)</td>
			<td></td>
			<td></td>
			<td>Dilute from 20X SSC, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td></td>
			<td></td>
			<td>100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Staining solution</td>
			<td></td>
			<td></td>
			<td>200 &#956;g/mL iodonitrotetrazolium chloride, 200 &#956;g/mL 5-Bromo-4-chloro-3-indolyl phosphate disodium salt, in staining buffer</td>
		</tr>
		<tr>
			<td>Stopping solution</td>
			<td></td>
			<td></td>
			<td>1 mM EDTA, pH 5.5, in PBST</td>
		</tr>
		<tr>
			<td>Torula (yeast) RNA</td>
			<td>Sigma-Aldrich</td>
			<td>R6625</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma Aldrich</td>
			<td>E10521</td>
			<td>2%, pH 7</td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td></td>
			<td></td>
			<td>50% formamide, 2X SSC, 0.1% Tween-20</td>
		</tr>
	</tbody>
</table>

in situ hybridization in zebrafish larvae and juveniles during mesonephros development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

Using the Tg(lhx1a-EGFP) transgenic line, it was demonstrated that this in situ hybridization protocol is effective in labeling kidney progenitor cells and various nephron structures during mesonephros development. As expected, the central nervous system is also labeled in this transgenic line (not shown). The initial mesonephric nephron forms at approximately 5.2 mm, dorsal to the pronephros (Figure 2A), and the distal tubule of this nephron is labeled by EGFP<sup class="x...

Watch this Scientific Journal Video about In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development at JoVE.com

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

This protocol is significant because it allows the determination of gene expression in older zebrafish larvae and juveniles during metamorphosis. The advantage of this technique is that several steps have been optimized for probe penetration and visualization of the kidney. To begin, set up adult zebrafish to mate by adding one male and one female fish in a mating tank in the late afternoon after their last meal.The next day, collect the embryos in Petri plates containing E3 medium. Five days post-fertilization, place a 400-micrometer screen in a 2.8-liter tank and fill it with 2 centimeters of system water. Then add the larva from one Petri plate.To fix the larva, remove a tank of larva at the desired time point, then, using a net and transfer pipette with its tip cut off, transfer the larva to the Petri plate. Next, add two milliliters of 2%tricaine to immobilize the larva. After immobilization, replace the tricaine with 20 milliliters of fixing solution.After 30 minutes, transfer the larva into a 50-milliliter tube containing 25 milliliters of fresh fixing solution. Then, ensuring that the cap is tight, slowly rock the tube at four degrees Celsius for two days. On day three, replace the fixing solution with 20 milliliters of PBST then transfer the larva to a Petri plate.To measure the larva, place the Petri plate on top of a flat ruler under a dissecting microscope, then, using an eyelash manipulator, move each larva onto the ruler to measure its total length. After measuring all larva, combine several larva of similar lengths onto one 5.5-milliliter glass vial with PBST. For dehydration, replace the PBST in the glass vial with four milliliters of 100%methanol, then store the vial at minus 20 degrees Celsius for two days.For rehydration, replace the 100%methanol with four milliliters of a 75%methanol and 25%PBST solution and rock the vial for five minutes. Then, replace the methanol and PBST solution with four milliliters of fresh PBST and rock the vial again for 10 minutes. For Proteinase K digestion, replace the PBST with two milliliters of a Proteinase K solution and rock the vial at room temperature for 30 minutes.Next, for bleaching, transfer the larva to a six-well plate and replace the PBST with three milliliters of fresh bleaching solution. After the complete disappearance of pigmentation along the mesonephros, transfer the larva back into a glass vial, replace the bleaching solution with four milliliters of PBST, and rock the vial for 10 minutes. For pre-hybridization, replace the fixing solution with four milliliters of PBST and rock the vial for 10 minutes, then, replace the PBST with four milliliters of Hyb-solution and rock for 10 minutes.After two additional incubations in the Hyb-solution, replace the solution with four milliliters of the Hyb+solution. Simultaneously dilute the EGFP fluorescein probe one to 100 in 500 microliters of hybridization plus solution, and incubate the vial and probe at 70 degrees Celsius overnight. The next day, to hybridize the probe, replace the Hyb+solution in the vial with the preheated probe and incubate at 70 degrees Celsius overnight.The following day, add 50 milliliters of preheated 0.2 times SSCT into a 50-milliliter tube. Then, insert a 100-micrometer cell strainer into the top of the tube, transfer the larva from the glass vial into the cell strainer, and ensuring that the larva are submerged in the buffer, incubate the vial at 70 degrees Celsius for two hours. After the incubation, transfer the cell strainer into a new tube containing preheated 0.2 times SSCT, and incubate again at 70 degrees Celsius for two hours.Next, for blocking, transfer the larva to a new glass vial and allow it to cool to room temperature. Then, replace the 0.2 times SSCT solution with four milliliters of a 67%0.2 times SSCT and 33%MABT solution and rock the vial at room temperature for 10 minutes. Next, replace the SSCTT MABT solution with four milliliters of fresh MABT, and incubate the vial on a rocker for 10 minutes.Then, replace the MABT with four milliliters of blocking solution and incubate four degrees Celsius overnight. The next day, replace the blocking solution with an antibody solution and incubate at four degree Celsius for two days. For antibody washing, transfer the larva into a 50-milliliter tube, then add 40 milliliters of PBST2, and lay the tube laying on its side for overnight incubation at four degrees Celsius.On day 12, after transferring the lava to a six-well plate, replace the PBST2 with three milliliters of staining buffer. After a five-minute incubation on a rocker, replace the staining buffer with three milliliters of the staining solution. When the desired staining intensity is reached, replace the staining solution with three milliliters of the stopping solution and rock for 30 minutes.Then, transfer the larva to a new glass vial, replace the stopping solution with four milliliters of fresh fixing solution, and incubate for one hour at room temperature. For imaging, replace the fixing solution with four milliliters of fresh PBST. Following a 10 minute incubation on a rocker, transfer the larva to a six-well plate, then replace the PBST with four milliliters of fresh PBST and rock the plate again for 10 minutes.Next, add three milliliters of 50%glycerol in PBST and rock for 10 minutes, then, using a dissecting microscope, image the larva directly in the six-well plate. This in situ hybridization protocol effectively labels kidney progenitor cells and various nephron structures using mesonephros development. The initial mesonephric nephron forms at approximately 5.2 millimeters dorsal to the pronephros.Clusters of progenitor cells are present during mesonephros development in addition to single progenitor cells. In larger juveniles, background staining can occur in the somites. Overall, this method can be used to study other tissues that form during metamorphosis, in addition to dissected adult organs.

Research

JoVE Journal

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...