This work was supported in part by the grant R01DK100237 to R.A.W. and the National Science Foundation Graduate Research Fellowship No. DGE-1313583 to A.N.M. We also thank the College of Science Summer Undergraduate Research Fellowship Program for support to M.U. We would like to thank the Center for Zebrafish Research at the University of Notre Dame for their dedicated care of our zebrafish. We would also like to thank the Department of Biological Sciences as well as the members of our lab for all of their support and valuable insights.

The following protocol uses zebrafish adults maintained and cared for by the Center for Zebrafish Research at the University of Notre Dame. All methods for working with zebrafish adults and embryos were approved by the Institutional Animal Care and Use Committee.
1. Embryo Fixation
<ol>
	<li>Collect zebrafish embryos using previously described methods39,40. At 24 hpf, add 500 &#181;L of non-specific protease mixture solution (50 mg/mL) to the E3 in an embryo dish Incubate at room temperature (RT).</li>
	<li>Once embryos begin breaking out of the chorion, remove all liquid from the embryo dish.</li>
	<li>Wash the embryos 2&#160;- 3 times with fresh E3 by adding approximately 20 mL of E3, gently swirling the E3 in the dish, and then decanting the fluid into a liquid waste receptacle.</li>
	<li>To euthanize the embryos before fixing, add&#160;2 mL of 0.2% tricaine to the E3.</li>
	<li>Transfer the embryos into a 5 mL glass vial using a plastic Pasteur pipette. Remove most of the tricaine/E3 solution using the pipette after the embryos have settled to the bottom of the vial.</li>
	<li>Fix 24 hpf embryos with 5 mL of 4% paraformaldehyde (PFA) for 2 - 4 h at RT, without agitation. 
	CAUTION: PFA is toxic and PFA solutions should be handled under a chemical hood while the researcher is wearing eye protection, gloves, and a lab coat. Preparation of the PFA fixative from the granulated PFA powder should be performed with exceptional care, as the granulated PFA tends to disperse through static charge. 
	NOTE: Embryos can fix in 4% PFA overnight at 4 &#176;C. Prepare 4% PFA by heating 1x phosphate buffered saline (PBS) to a boil while continually stirring the solution to completely dissolve the granulated PFA. Once the solution has cooled back down to RT, the 4% PFA is filter sterilized by passing it through a 0.45 &#181;m disposable system for vacuum filtration, then and aliquoted into 15 mL or 50 mL portions for storage at -20 &#176;C.</li>
	<li>Remove the 4% PFA and wash embryos twice with 1X phosphate buffered saline with 0.1% Tween-20 (PBST) at RT. Wash the embryos once with 5 mL of 100% methanol (MeOH) at RT. Add 5 mL of fresh 100% MeOH to the embryos and incubate at -20 &#176;C for at least 20 min. 
	NOTE: Embryos can be stored in 100% MeOH at -20&#176;C until ready for embryo preparation.</li>
</ol>
2. Embryo preparation and hybridization
<ol>
	<li>Rehydrate the embryos by washing once in 5 mL of 50% MeOH in 1x PBST for 5 min at RT. Continue rehydration by washing the embryos in 5 mL of 30% MeOH in 1x PBST for 5 min at RT. Wash the embryos in 5 mL of 1x PBST twice for 5 min each at RT.</li>
	<li>Permeabilize the embryos by incubating them in 5 mL of a solution of 1:1,000 proteinase K (pK) (10 mg/mL) in 1x PBST for 2 min at RT. 
	NOTE: Older embryos can be incubated longer than 2 min in the pK concentration stated above, however a concentration of 5 mg/mL and incubation time less than 2 min is suggested for embryos younger than 24 hpf.</li>
	<li>Remove pK solution and wash immediately in 5 mL of 1x PBST twice at RT. Fix the embryos in 5 mL of 4% PFA at RT for at least 20 min. 
	NOTE: Embryos can fix in 4% PFA overnight at 4 &#176;C.</li>
	<li>Remove PFA and wash twice in 5 mL of 1x PBST at RT.</li>
	<li>Transfer embryos in 5 mL of 1x PBST into flat bottom microcentrifuge tubes placed upright into a microcentrifuge rack at RT to facilitate interactions between each embryo and the subsequent hybridization solution.</li>
	<li>Wash the embryos twice in 1.5 mL of hybridization solution (Hyb+) at RT. Add 1.5 mL of fresh Hyb+ and incubate embryos at 70 &#176;C in a hybridization oven for 4-6 h.</li>
	<li>Replace the 1.5 mL of Hyb+ with a volume of probe solution (10 &#181;L RNA probe/500 &#181;L Hyb+) sufficient to fully cover the embryos. 
	NOTE: Synthesize antisense RNA probe using previously described methods39.</li>
	<li>Hybridize the probe overnight in the hybridization oven at 70 &#176;C with the individual tubes positioned upright in the microcentrifuge rack, without shaking.</li>
</ol>
3. Hot Washes and Blocking
NOTE: Hot washes are performed by putting the appropriate solution on the embryos and then incubating in the hybridization oven at 70 &#176;C. To keep the washing solutions at 70 &#176;C, place 50 mL tubes of each solution in the hybridization oven when the probe/Hyb+ mixture is added.
<ol>
	<li>Remove the probe. Wash the embryos twice at 70 &#176;C in 1.5 mL of 50% formamide/2x saline-sodium citrate (SSC) buffer for 20-30 min each. 
	NOTE: The probe can be stored in Hyb+ at -20 &#176;C and re-used at a later date.</li>
	<li>Wash the embryos once at 70 &#176;C in 1.5 mL of 2x SSC for 15 min. Wash the embryos twice at 70 &#176;C in 1.5 mL of 0.2X SSC for 20-30 min each. Replace 0.2x SSC with 1.5 mL of blocking reagent and incubate overnight at 4 &#176;C. 
	NOTE: It is possible to incubate blocking reagent at RT for 4 h instead of overnight.</li>
</ol>
4. Antibody Incubation and Maleic Acid Buffer Washes
NOTE: Keep embryos protected from ambient light.
<ol>
	<li>Replace the blocking reagent with 0.2 mL of anti-DIG-POD in blocking reagent at a ratio of 1:1,000 and incubate for 3 h under foil or another light-blocking cover at RT. 
	NOTE: Alternatively, the antibody can incubate overnight at 4 &#176;C.</li>
	<li>After 3 h, wash the embryos in 1.5 mL of maleic acid buffer 2-4 times for 10-15 min each at RT. Incubate the embryos overnight at 4 &#176;C in 1.5 mL of fresh maleic acid buffer. 
	NOTE: It is not necessary to incubate in maleic acid buffer overnight, but doing so decreases probe background.</li>
</ol>
5. Probe Detection and Removal of Peroxidase
NOTE: Keep embryos protected from ambient light.
<ol>
	<li>Remove maleic acid buffer and replace with 1.5 mL of 1x PBS. Wash embryos twice in 1.5 mL of 1x PBS for 5 min each at RT. Incubate embryos in 0.2 mL of Cy3 fluorescent staining solution for 60 min at RT. 
	NOTE: Incubation time may vary for different probes.</li>
	<li>Wash the embryos once with 1.5 mL of the following percent MeOH in 1X PBS for 10 min at RT: 30% MeOH, 50% MeOH, 75% MeOH and 100% MeOH.</li>
	<li>Incubate the embryos with 1.5 mL of 1% H2O2 in MeOH for 30 min at RT. Wash the embryos once with 1.5 mL of the following MeOH solutions in 1x PBS for 10 min at RT: 75% MeOH, 50% MeOH, and 30% MeOH. Wash the embryos twice for 5 min each at RT in 1.5 mL of 1x PBS. 
	NOTE: Embryos can be stored in PBS overnight at 4&#176;C.</li>
</ol>
6. Immunofluorescence
NOTE: Keep embryos protected from ambient light.
<ol>
	<li>Wash the embryos in 1.5 mL of ddH2O for 5 min at RT. Wash the embryos in 1.5 mL of pre-chilled acetone (kept at -20 &#176;C) for 7 min at RT. Wash the embryos once in 1.5 mL of ddH2O for 5 min at RT. Wash the embryos once in 1.5 mL of 1x PBST with 1% DMSO (PBDT) for 5 min at RT.</li>
	<li>Incubate the embryos in 1.5 mL of PBDT + 10% fetal bovine serum (FBS) on a rocker at RT for 2 h.</li>
	<li>Remove PBDT + FBS and replace with 1.5 mL of primary antibodies, anti-acetylated tubulin (produced from mouse) and anti-&#947;-tubulin (produced from rabbit), diluted together at 1:400 in PBDT + 1% FBS. Incubate the embryos overnight at 4 &#176;C. 
	NOTE: Incubation times may vary depending on the primary antibody. Testing of different time intervals may be necessary to optimize labeling.</li>
</ol>
7. Secondary Antibody
NOTE: Keep embryos protected from ambient light.
<ol>
	<li>To remove excess unbound antibody from the embryos, wash in 1.5 mL of PBDT + 1% FBS + 0.1 M NaCl for 1 min at RT on a rocker.</li>
	<li>Wash the embryos 5 times in 1.5 mL of PBDT + 1% FBS + 0.1 M NaCl for 30 min each on a rocker at RT. Wash the embryos in 1.5 mL of PBDT + 1% FBS for 30 min at RT on a rocker.</li>
	<li>Incubate the embryos overnight at 4 &#176;C in 200 &#181;L of the secondary antibodies, Alexa 488 goat anti-mouse IgG and Alexa 647 goat anti-rabbit IgG, diluted together in PBDT at 1:500.</li>
</ol>
8. DAPI Staining
NOTE: Keep embryos protected from ambient light.
<ol>
	<li>Wash the embryos quickly at RT in 1.5 mL of PBDT two times. Replace PBDT with 1.5 mL of DAPI, diluted 1:15000 in 1X PBST, and incubate 15 min at RT on a rocker.</li>
	<li>Wash the embryos in 1.5 mL of PDBT + 1% FBS + 0.1 M NaCl three times at RT for 15-20 min each. Store embryos in 1.5 mL of PBDT at 4 &#176;C until ready to mount and image.</li>
</ol>
9. Mounting and Imaging for Zebrafish Pronephros
<ol>
	<li>Lay embryo lateral on glass slide in a small volume, approximately 10 &#181;L, of PBDT.</li>
	<li>Squeeze the embryo behind the eyes with a pair of fine forceps to remove the head and some of the yolk ball.</li>
	<li>Use the fine forceps to gently scrape away the remaining yolk ball from the embryo body. Remove the dissociated yolk and extra liquid from the slide with a thin tissue.</li>
	<li>Add a drop of mounting media to the remaining tail of the embryo and position so the tail is laid laterally. Place a coverslip on top of the tail to flatten the sample, then place in a covered slide box.</li>
	<li>Image kidney cilia at 60X magnification on a confocal microscope using the following channels: DAPI in blue (laser set at 408.0 nm), FITC in green (laser set at 488.0 nm), Cy3 dye-labeled in red (laser set at 561.0 nm), and Alexa 680 in white (laser set at 637.0 nm).</li>
</ol>

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8, 353-367 (2007).</li><li>Goldsmith, J. R., Jobin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Think+small:+zebrafish+as+a+model+system+of+human+pathology.">Think small: zebrafish as a model system of human pathology.</a> J Biomed Biotechnol. 2012, 817341 (2012).</li><li>Howe, K., 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496, 498-503 (2013).</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> J Am Soc Nephrol. 16, 299-304 (2005).</li><li>Wingert, R. 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=The+cdx+genes+and+retinoic+acid+control+the+positioning+and+segmentation+of+the+zebrafish+pronephros.">The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.</a> PLoS Genet. 3, 1922-1938 (2007).</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+segmentation.">The zebrafish pronephros: a model to study segmentation.</a> Kindey Int. 73 (10), 1120-1127 (2008).</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=Zebrafish+nephrogenesis+involves+dynamic+spatiotemporal+expression+changes+in+renal+progenitors+and+essential+signals+from+retinoic+acid+and+irx3b.">Zebrafish nephrogenesis involves dynamic spatiotemporal expression changes in renal progenitors and essential signals from retinoic acid and irx3b.</a> Dev Dyn. 240, 2011-2027 (2011).</li><li>Chambers, B. E., 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=Renal+progenitors:+roles+in+kidney+disease+and+regeneration.">Renal progenitors: roles in kidney disease and regeneration.</a> World J Stem Cells. 8 (11), 367-375 (2016).</li><li>Drummond, B. E., 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=Insights+into+kidney+stem+cell+development+and+regeneration+using+zebrafish.">Insights into kidney stem cell development and regeneration using zebrafish.</a> World J Stem Cells. 8 (2), 22-31 (2016).</li><li>Poureetezadi, S. J., 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=Little+fish,+big+catch:+zebrafish+as+a+model+for+kidney+disease.">Little fish, big catch: zebrafish as a model for kidney disease.</a> Kidney Int. 89 (6), 1204-1210 (2016).</li><li>Kramer-Zucker, A. G., Olale, F., Haycraft, C. J., Yoder, B. K., Schier, A. F., 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=Cilia-driven+fluid+flow+in+the+zebrafish+pronephros,+brain+and+Kupffer's+vesicle+is+required+for+normal+organogenesis.">Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis.</a> Development. 132, 1907-1921 (2005).</li><li>Liu, Y., Narendra, P., 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+epithlelia+and+multiciliated+cells+in+the+zebrafish+pronephros.">Notch signaling controls the differentiation of transporting epithlelia and multiciliated cells in the zebrafish pronephros.</a> Development. 134, 1111-1122 (2007).</li><li>Ma, M., Jiang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jagged2a-Notch+signaling+mediates+cell+fate+choice+in+the+zebrafish+pronephric+duct.">Jagged2a-Notch signaling mediates cell fate choice in the zebrafish pronephric duct.</a> PLoS Genetics. 3, e18 (2007).</li><li>Li, Y., Cheng, C. N., Verdun, V. A., 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=Zebrafish+nephrogenesis+is+regulated+by+interactions+between+retinoic+acid,+mecom,+and+Notch+signaling.">Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and Notch signaling.</a> Dev Biol. 386 (1), 111-122 (2014).</li><li>Wang, L., 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=miR-34b+regulates+multiciliogenesis+during+organ+formation+in+zebrafish.">miR-34b regulates multiciliogenesis during organ formation in zebrafish.</a> Development. 140, 2755-2764 (2013).</li><li>Marra, A. 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=Epithelial+cell+fate+in+the+nephron+tubule+is+mediated+by+the+ETS+transcription+factors+etv5a+and+etv4+during+zebrafish+development.">Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish development.</a> Dev Biol. 411 (2), 231-245 (2016).</li><li>Marra, A. N., Li, Y., 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=Antennas+of+organ+morphogenesis:+the+roles+of+cilia+in+vertebrate+kidney+development.">Antennas of organ morphogenesis: the roles of cilia in vertebrate kidney development.</a> Genesis. 54 (9), 457-469 (2016).</li><li>Duffy, J. L., Suzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliated+human+renal+proximal+tubular+cells.+Observations+in+three+cases+of+hypercalcemia.">Ciliated human renal proximal tubular cells. Observations in three cases of hypercalcemia.</a> Am J Pathol. 53, 609-616 (1968).</li><li>Katz, S. M., Morgan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia+in+the+human+kidney.">Cilia in the human kidney.</a> Ultrastruct Pathol. 6, 285-294 (1984).</li><li>Hassan, M. O., Subramanyan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliated+renal+tubular+cells+in+crescentic+glomerulonephritis.">Ciliated renal tubular cells in crescentic glomerulonephritis.</a> Ultrastruct Pathol. 19, 201-203 (1995).</li><li>Ong, A. C., Wagner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+proximal+tubular+motile+cilia+in+a+patient+with+renal+sarcoidosis+associated+with+hypercalcemia.">Detection of proximal tubular motile cilia in a patient with renal sarcoidosis associated with hypercalcemia.</a> Am J Kidney Dis. 45, 1096-1099 (2005).</li><li>Worthington, W. C., Cathcart, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ependymal+cilia:+distribution+and+activity+in+the+adult+human+brain.">Ependymal cilia: distribution and activity in the adult human brain.</a> Science. 139, 221-222 (1963).</li><li>Cowan, M. J., Galdwin, M. T., Shelhamer, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+ciliary+motility.">Disorders of ciliary motility.</a> Am J Med Sci. 321, 3-10 (2001).</li><li>Jain, 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=Temporal+relationship+between+primary+and+motile+ciliogenesis+in+airway+epithelial+cells.">Temporal relationship between primary and motile ciliogenesis in airway epithelial cells.</a> Am J Respir Cell Mol Biol. 43, 731-739 (2010).</li><li>Stubbs, J. L., Vladar, E. K., Axlerod, J. D., Kitner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicilin+promotes+centriole+assembly+and+ciliogenesis+during+multiciliate+cell+differentiation.">Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation.</a> Nat Cell Biol. 14, 140-147 (2012).</li><li>Tan, F. E., 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=Myb+promotes+centriole+amplification+and+later+steps+of+the+multiciliogenesis+program.">Myb promotes centriole amplification and later steps of the multiciliogenesis program.</a> Development. 140, 4277-4286 (2013).</li><li>Zhou, F., Narasimhan, V., Shboul, M., Chong, Y. L., Reversade, B., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gmnc+is+a+master+regulator+of+the+multiciliated+cell+differentiation+program.">Gmnc is a master regulator of the multiciliated cell differentiation program.</a> Curr Biol. 25, 3267-3273 (2015).</li><li>Jowett, T. <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+Protein+and+gene+expression.">Analysis of Protein and gene expression.</a> Methods Cell Biol. 59, 63-85 (1999).</li><li>Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2:+Looking+at+Embryos.">Chapter 2: Looking at Embryos.</a> Zebrafish: A practical approach. 261, 39-58 (2002).</li><li>Brend, T., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+whole+mount+high-resolution+double+fluorescent+in+situ+hybridization.">Zebrafish whole mount high-resolution double fluorescent in situ hybridization.</a> J Vis Exp. (25), (2009).</li><li>Lauter, G., Soll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+fluorescent+in+situ+hybridization+in+the+embryonic+zebrafish+brain+using+differential+detection+systems.">Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.</a> BMC Dev Biol. 11, (2011).</li><li>Drummond, B. E., Li, Y., Marra, A. N., Cheng, C. 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=The+tbx2a/b+transcription+factors+direct+pronephros+segmentation+and+corpuscle+of+Stannius+formation+in+zebrafish.">The tbx2a/b transcription factors direct pronephros segmentation and corpuscle of Stannius formation in zebrafish.</a> Dev Biol. 421 (1), 52-66 (2017).</li><li>Jaffe, K. M., Thiberge, S. Y., Bisher, M. E., Burdine, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cilia+in+zebrafish.">Imaging cilia in zebrafish.</a> Methods Cell Biol. 97, 415-435 (2010).</li><li>Lauter, G., Soll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+fluorescent+in+situ+hybridization+to+define+abutting+and+overlapping+gene+expression+in+the+embryonic+zebrafish+brain.">Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.</a> Neural Dev. 6, (2011).</li><li>Sorrells, S., Toruno, C., Stewart, R. A., Jette, C. <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+apoptosis+in+zebrafish+embryos+by+whole-mount+immunofluorescence+to+detect+activated+caspase+3.">Analysis of apoptosis in zebrafish embryos by whole-mount immunofluorescence to detect activated caspase 3.</a> J Vis Exp. (82), e51060 (2013).</li><li>Schumacher, J. A., Zhao, E. J., Kofron, M. J., Sumanas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+fluorescent+in+situ+hybridization+using+chromogenic+substrates+in+zebrafish.">Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish.</a> Bio Techniques. 57, 254-256 (2014).</li><li>Julich, D., 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=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Gross-Thebing, T., Paksa, A., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+detection+of+multiple+transcripts+combined+with+localization+of+proteins+in+whole-mount+embryos.">Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos.</a> BMC Biology. 12, (2014).</li><li>Cheng, C. N., Li, Y., Marra, A. N., Verdun, V., 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=Flat+Mount+Preparation+for+Observation+and+Analysis+of+Zebrafish+Embryo+Specimens+Stained+by+Whole+Mount+In+situ+Hybridization.">Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization.</a> J Vis Exp. (89), e51604 (2014).</li><li>Poureetezadi, S. J., Donahue, E. K., 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=A+Manual+Small+Molecule+Screen+Approaching+High-throughput+Using+Zebrafish+Embryos.">A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos.</a> J Vis Exp. (93), e52063 (2014).</li></ol>

The authors have nothing to disclose.

The protocol detailed above is optimized for labeling MCCs in the pronephros with odf3b transcripts and &#945;-tubulin in 24 hpf zebrafish embryos. For the best results, it is recommended to use freshly fixed and prepared embryos. Embryos that have been fixed and stored in either MeOH or Hyb+ at -20 &#176;C for longer than one week can be used, but the probability of unwanted background staining increases with the time that the embryos are kept in storage.
Modifications and troubleshooting of this technique are necessary to tailor this method for other suites of particular markers, e.g. other antisense riboprobes and other antibodies. Many methods for adjusting the parameters of steps in the process of whole mount in situ hybridization have been previously documented by our group and others31,34,36,38,39. Likewise, each protein antibody will require some troubleshooting in terms of staining times, and approximate ranges of dilutions and incubation times for each primary antibody are best estimated by evaluation of documented results28,35. For embryos younger than 24 hpf, pK treatment should be shorter than 2 min, where embryos older than 24 hpf should be treated with pK for longer than 2 min. Also, embryos older than 24 hpf should be bleached of pigment before pK treatment.
After staining with the fluorescent staining solution, it is critical to remove any remaining stain, antibody, and peroxidases by performing the series of methanol and hydrogen peroxide washes. The PBS washes immediately following are vital to remove excess methanol from the embryos. Importantly, before proceeding with the IF antibodies, we have found that the acetone and deionized water washes make the embryos less likely to adhere to one another and/or the centrifuge tubes. In our experience, antibodies for specific transcription factors and other genes, though they may work efficiently in Western blots, do not work well for IF in the zebrafish. However, highly conserved, and abundant proteins, such as &#945;-tubulin and &#946;-catenin, do work well in the zebrafish for IF16,37.
With FISH, it is possible to visualize RNA transcripts of genes that do not yet have specific antibodies in the zebrafish. By combining FISH with IF, as demonstrated by this protocol, co-localization of RNA transcripts and protein can be seen in vivo (Figure 2). The flexible nature of the protocol enables quick troubleshooting for the visualization of various RNA transcripts and proteins in numerous tissues and time points. When combined with the already respected traits of zebrafish embryos, this protocol of FISH + IF provides another tool to explore the expression of genes and proteins important to developmental pathway regulation.

Over the past several decades, the zebrafish (Danio rerio) has emerged as a prime model organism to study developmental biology. The embryos develop outside of the mother and are optically transparent. Also, the formation of vital organs such as the eye, kidney, and forebrain occurs rapidly, with structures formed by just 24 h post fertilization (hpf). Importantly, the zebrafish genome is highly conserved with mammals1,2,3. Additionally, zebrafish and mammalian organs have similar anatomy and physiology. The zebrafish embryonic kidney, or pronephros, demonstrates the value of the model system for examining gene function during early nephrogenesis and fate determination of conserved epithelial cell populations of the vertebrate nephron4,5,6,7,8,9,10. Similarly, the zebrafish embryo has become increasingly important in examining the ontogeny of MCCs11,12,13,14,15,16,17.
As their name suggests, MCCs are epithelial cells characterized by a bundle of motile cilia located on the apical surface17. In the zebrafish, MCCs function in fluid flow and are dispersed in a &#34;salt-and-pepper&#34; like fashion throughout the middle of each nephron of the pronephros by 24 hpf11,12,13,14,15,16,17. Although they have only been noted in a handful of human kidney disease cases18,19,20,21, MCCs are prevalent in other mammalian tissues such as the brain and trachea22,23,24, which poses a host of challenges for experimental design. Elegant studies in various vertebrate models including zebrafish have demonstrated a conserved pathway of MCC fate, with the Notch signaling pathway as an inhibitor of MCC development12,17,25,26,27. Therefore, the zebrafish pronephros provides an easily accessible model to study the genetic mechanisms of MCC development in vivo11,12,13,14,15,16,17.
Both transparency and easy genetic manipulation of zebrafish embryos have proven to be invaluable traits when studying the genetic and molecular pathways that regulate cell fate, tissue growth, and development of the early embryo1,2,3. As such, traditional techniques to visualize protein and gene transcripts, such as in situ hybridization and whole mount IF, have been applied to and optimized for the zebrafish16,28,29,30,31,32,33,34,35,36,37,38. By combining popular protocols for FISH and IF, it is possible to label and analyze MCCs in vivo16,28,37,38.

<table>
	<tbody>
		<tr>
			<td>non-specific protease mixture solution</td>
			<td>Roche</td>
			<td>11459643001, pronase from Streptomyces griseus</td>
			<td>Dilute in E3 without methylene blue to make 50mg/mL stock solution; store at -20&#176;C</td>
		</tr>
		<tr>
			<td>E3 solution</td>
			<td></td>
			<td></td>
			<td>Dilute 50X E3 (250 mM NaCl, 8.5 mM KCl, 16.5 mM CaCl2, 16.5 mM MgSO4 in distilled water) in distilled water; add 1 x 10-5 M methylene blue (sigma M9140) to inhibit contamination</td>
		</tr>
		<tr>
			<td>tricaine (ethyl 3-aminobenzoate methanesulfonate)</td>
			<td>Fluka</td>
			<td>A5040-250G</td>
			<td>To make a 0.2% stock solution, dissolve 1 g of tricaine powder in 10 mL Tris, pH 9.5 and distilled water up to 500 mL</td>
		</tr>
		<tr>
			<td>embryo dishes</td>
			<td>VWR</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL glass vials</td>
			<td>Wheaton</td>
			<td>225012</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable plastic Pasteur pippettes</td>
			<td>VWR</td>
			<td>414004-004</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde (PFA) solution</td>
			<td>Electron Microscopy Services</td>
			<td>19210</td>
			<td>Dissolve 4% PFA in 1X PBS and bring to a boil under a fume hood. Cool and aliquot, then store at -20&#176;C. Do not refreeze once thawed for use.</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>American Bioanalytical</td>
			<td>AB11072</td>
			<td>Dilute in distilled water to make a 1X stock</td>
		</tr>
		<tr>
			<td>Tween-20 stock</td>
			<td>American Bioanalytical</td>
			<td>AB02038</td>
			<td>Make a 0.1% Tween-20 stock by diluting in distilled water.</td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline with 0.1% Tween 20 (PBST)</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td>0.1% Tween-20 in 1X PBS</td>
		</tr>
		<tr>
			<td>methanol (MeOH)</td>
			<td>Sigma</td>
			<td>34860-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td>Dissolve in distilled water to make a 10 mg/mL stock; aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>flat bottom microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-300; 87003-298</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>American Bioanalytical</td>
			<td>AB00600</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>hybridization solution (HYB+)</td>
			<td></td>
			<td></td>
			<td>50% formamide, 5X SSC, 0.1% Tween-20, 5 mg/mL yeast torula RNA, 50 &#956;g/&#956;L heparin; store at -20&#176;C</td>
		</tr>
		<tr>
			<td>hybridization oven</td>
			<td>Fisher Scientific</td>
			<td>13-247-10Q</td>
			<td></td>
		</tr>
		<tr>
			<td>20X saline-sodium citrate (SSC) buffer solution</td>
			<td>American Bioanalytical</td>
			<td>AB13156</td>
			<td>dilute in distilled water to make 2X and 0.2X stocks</td>
		</tr>
		<tr>
			<td>blocking reagent solution</td>
			<td>Roche</td>
			<td>11096176001</td>
			<td>dilute in maleic acid buffer to make a 10% stock solution; store at 4&#176;C</td>
		</tr>
		<tr>
			<td>maleic acid buffer solution</td>
			<td>Sigma</td>
			<td>M0375 and S7653</td>
			<td>150mM maleic acid, 100mM NaCl (pH 7.5)</td>
		</tr>
		<tr>
			<td>anti-Digoxigenin-POD, Fab fragments</td>
			<td>Roche</td>
			<td>11207739910</td>
			<td>store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Cy3 fluorescent staining solution</td>
			<td>PerkinElmer, Inc.</td>
			<td>NEL744001KT, TSA Plus Cyanin3 system</td>
			<td>store at 4&#176;C; prepare staining solution fresh by making a 1:50 dilution of TSA reagent (dissolved in 60 uL of DMSO) in the kit buffer</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2)</td>
			<td>Sigma</td>
			<td>H1009-500mL</td>
			<td>store at 4&#176;C</td>
		</tr>
		<tr>
			<td>molecular grade distilled water (ddH2O)</td>
			<td>Mediatech</td>
			<td>25-055-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>American Bioanalytical</td>
			<td>AB00636-01000</td>
			<td>store an aliquot at -20&#176;C</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>American Bioanalytical</td>
			<td>AB00435-01000</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10438-034</td>
			<td>aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>monoclonal anti-acetylated tubulin clone 6-11B-1</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td>aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>anti-&#947;-tubulin anitbody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>T5192</td>
			<td>aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>odf3b cDNA clone MGC:63985</td>
			<td>OpenBiosystems</td>
			<td>IMAGE:6792478</td>
			<td>store bacterial glycerol stock at -80&#176;C</td>
		</tr>
		<tr>
			<td>NaCL</td>
			<td>American Bioanalytical</td>
			<td>AB01915-05000</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa flour 647 goat anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A21245</td>
			<td>store at 4&#176;C; protect from light</td>
		</tr>
		<tr>
			<td>Alexa fluor 488 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11029</td>
			<td>store at 4&#176;C; protect from light</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td>aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>glass slide</td>
			<td>Thermo-Fisher</td>
			<td>4445</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslip</td>
			<td>Thermo-Fisher</td>
			<td>12-540A</td>
			<td>18 x 18 mm</td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Roboz</td>
			<td>RS-1050</td>
			<td>Dumont Tweezers Pattern #55</td>
		</tr>
		<tr>
			<td>mounting media</td>
			<td>Polysciences, Inc.</td>
			<td>18606, Aqua-Poly/Mount</td>
			<td>store at 4&#176;C</td>
		</tr>
		<tr>
			<td>confocal microscope and associated software</td>
			<td></td>
			<td></td>
			<td>We use a Nikon C2plus Confocal Microscope with NIS-elements AR software</td>
		</tr>
		<tr>
			<td>rocker</td>
			<td>Bio-Rad</td>
			<td>1660710EDU</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Wild-type zebrafish embryos were fixed at 24 hpf and immediately prepared as described above. Figure 1 depicts an experimental workflow along with selected illustrated stages. The workflow outlined in Steps 1-8 encompasses the processes of sample procurement, fixation, and manipulation of the fixed tissue to label endogenous transcripts with antisense riboprobes followed by immunofluorescence to label protein(s) of interest. Step 9 in the workflow refers to the mounting and imaging techniques specifically designed to optimize visualization of the zebrafish embryonic trunk. In the drawings that accompany Step 9, we illustrate the method of manipulation for positioning the tissue on a glass slide. In this step, the embryo head and yolk ball are removed, leaving the tail to be laterally positioned between a glass slide and coverslip. Removal of the yolk ball and head is suggested for the best imaging of the resident MCC population in the pronephros, as the yolk ball auto-fluoresces and obstructs the mounting process.
Representative images obtained using a confocal microscope are provided in Figure 2, which shows the same wild-type embryo at 60X magnification and a digital zoom at the same magnification. The top panels provide images of each individual channel, while the bottom two panels are the composite overlay of these imaging data. The white box (in the left column image) outlines the area that is the focus of the digital zoom (the right column image). Highlighted in the final panel of the digital zoom, nuclei were labeled in DAPI, and MCCs were detected based on an antisense riboprobe designed to recognize odf3b, where antibodies to &#947;-tubulin denote the basal bodies and &#945;-tubulin denotes the cilia. In the digital zoom of the composite overlay, the yellow dashed circle indicates the perimeter of an MCC, which was identified due to the possession of odf3b transcripts, multiple basal bodies, and cilia. The adjacent mono-ciliated cell, labeled with a yellow dotted circle, was identified based on the phenotype of possessing a single basal body and a single cilium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56261/56261fig1.jpg" /> 
Figure 1: Schematic of experimental flowchart. This flowchart shows an experimental workflow, accompanied by illustrations of critical stages in which the snowflake indicates cold incubation, the three curved lines represent heat, and the clock represents long incubation times. The workflow can be performed in a minimum of 6 days (see day number in upper right corner of each stage in the process), though some steps can be performed over longer time periods, as noted in the protocol. A detailed depiction of the mounting process is drawn out, demonstrating the final mounted embryo tail between the glass slide and coverslip. A black dashed box outlines the area that is imaged at 60X magnification. <a href="https://cloudflare.jove.com/files/ftp_upload/56261/56261fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56261/56261fig2.jpg" /> 
Figure 2: Representative results for visualizing MCCs of the zebrafish pronephros. Maximum image projections of a 24 hpf wild-type zebrafish embryo at 60X magnification as well as a digital zoom on the confocal at 60X magnification of the same embryo. The white boxes indicate the area focused on for the zoom. Individual stains of DAPI (nuclei), odf3b (MCCs), &#947;-tubulin (basal bodies), and &#945;-tubulin (cilia) are labeled and then merged together in the bottom two panels. In the digital zoom, we provide approximations of the cell locations as follows: an MCC is outlined by the dashed yellow circle, and the dotted yellow circle outlines a mono-ciliated cell. <a href="https://cloudflare.jove.com/files/ftp_upload/56261/56261fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence at JoVE.c

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

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

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 ...

visualizing-multiciliated-cells-zebrafish-through-combined-protocol

Research

JoVE Journal

Developmental Biology

8.1K Views.  University of Notre Dame. Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

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

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 is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

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

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

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

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

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 developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

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 zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

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. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

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