Name: whole mount immunohistochemistry in zebrafish embryos and larvae
Uploaded: 2020-01-29T14:30:00
Description: Watch this Scientific Journal Video about Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae at JoVE.com

Funding from NIH grant 8P20GM103436 14.

The procedures for working with zebrafish breeding adults and embryos described in this protocol were approved by the Institutional Animal Care and Use Committee at Murray State University.
1. Embryo Collection and Fixation
<ol>
	<li>Prepare spawning tanks by placing adult zebrafish mixed sex pairs or groups in tanks with a mesh or slotted liner filled with system water overnight.</li>
	<li>At lights on, change the spawning tank water for fresh system water to remove feces. Use a 14 h/10 h l...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+and+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (2013).</li><li>Irion, U., Krauss, J., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+and+efficient+genome+editing+in+zebrafish+using+the+CRISPR/Cas9+system.">Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system.</a> Development. 141 (24), 4827-4830 (2014).</li><li>van der Ploeg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+nucleic+acid+research+during+the+twentieth+century.">Cytochemical nucleic acid research during the twentieth century.</a> European Journal of Histochemistry. 44 (1), 7-42 (2000).</li><li>Marrack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+Antibodies.">Nature of Antibodies.</a> Nature. 133 (3356), 292-293 (1934).</li><li>Coons, A. H., Creech, H. J., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+properties+of+an+antibody+containing+a+fluorescent+group.">Immunological properties of an antibody containing a fluorescent group.</a> Proceedings of the Society for Experimental Biology and Medicine. 47 (2), 200-202 (1941).</li><li>Lopez, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+in+situ+Hybridization/Immunohistochemistry+(ISH/IH)+on+Free-floating+Vibratome+Tissue+Sections.">Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections.</a> Bio-protocol. 4 (18), (2014).</li><li>Morimoto, M., Morita, N., Ozawa, H., Yokoyama, K., Kawata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+glucocorticoid+receptor+immunoreactivity+and+mRNA+in+the+rat+brain:+an+immunohistochemical+and+in+situ+hybridization+study.">Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study.</a> Neuroscience Research. 26 (3), 235-269 (1996).</li><li>Newton, S. S., Dow, A., Terwilliger, R., Duman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+combined+immunohistochemistry+and+in-situ+hybridization+in+fresh-frozen,+cryocut+mouse+brain+sections.">A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen, cryocut mouse brain sections.</a> Brain Research Protocols. 9 (3), 214-219 (2002).</li><li>Corthell, J. T., Corthell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls. , 105-111 (2014).</li><li>Corthell, J. T., Corthell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls. , 91-103 (2014).</li><li>Vogel, C., Marcotte, E. M. <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+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nature Reviews Genetics. 13 (4), 227-232 (2012).</li><li>Semache, M., Ghislain, J., Zarrouki, B., Tremblay, C., Poitout, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+and+duodenal+homeobox-1+nuclear+localization+is+regulated+by+glucose+in+dispersed+rat+islets+but+not+in+insulin-secreting+cell+lines.">Pancreatic and duodenal homeobox-1 nuclear localization is regulated by glucose in dispersed rat islets but not in insulin-secreting cell lines.</a> Islets. 6 (4), e982376 (2014).</li><li>Kang, H. S., Beak, J. Y., Kim, Y. S., Herbert, R., Jetten, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glis3+is+associated+with+primary+cilia+and+Wwtr1/TAZ+and+implicated+in+polycystic+kidney+disease.">Glis3 is associated with primary cilia and Wwtr1/TAZ and implicated in polycystic kidney disease.</a> Molecular and Cellular Biology. 29 (10), 2556-2569 (2009).</li><li>Billova, S., Galanopoulou, A. S., Seidah, N. G., Qiu, X., Kumar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+expression+and+colocalization+of+somatostatin,+carboxypeptidase-E+and+prohormone+convertases+1+and+2+in+rat+brain.">Immunohistochemical expression and colocalization of somatostatin, carboxypeptidase-E and prohormone convertases 1 and 2 in rat brain.</a> Neuroscience. 147 (2), 403-418 (2007).</li><li>Westerfield, M. <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 Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2000).</li><li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: A Practical Approach. , (2002).</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> Journal of Visualized Experiments. (89), (2014).</li><li>Brennan, C., Mangoli, M., Dyer, C. E. F., Ashworth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+and+calcium+signalling+regulates+muscle+fibre+formation+in+the+zebrafish+embryo.">Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo.</a> Journal of Cell Science. 118 (22), 5181 (2005).</li><li>Liu, D. W., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustering+of+muscle+acetylcholine+receptors+requires+motoneurons+in+live+embryos,+but+not+in+cell+culture.">Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture.</a> The Journal of Neuroscience. 12 (5), 1859 (1992).</li><li>Borodinsky, L. N., Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+neurotransmitter-receptor+matching+at+the+neuromuscular+junction.">Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (1), 335-340 (2007).</li><li>Brunelli, G., 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=Glutamatergic+reinnervation+through+peripheral+nerve+graft+dictates+assembly+of+glutamatergic+synapses+at+rat+skeletal+muscle.">Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (24), 8752-8757 (2005).</li><li>Hans, F., Dimitrov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3+phosphorylation+and+cell+division.">Histone H3 phosphorylation and cell division.</a> Oncogene. 20 (24), 3021-3027 (2001).</li><li>Yee, N. S., 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=Zebrafish+as+a+model+for+pancreatic+cancer+research.">Zebrafish as a model for pancreatic cancer research.</a> Methods in Molecular Medicine. 103, 273-298 (2005).</li><li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+use+of+zebrafish+to+model+metabolic+disease.">The emerging use of zebrafish to model metabolic disease.</a> Disease Models &amp; Mechanisms. 6 (5), 1080-1088 (2013).</li><li>Fontana, B. D., Mezzomo, N. J., Kalueff, A. V., Rosemberg, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+utility+of+zebrafish+models+of+neurological+and+neuropsychiatric+disorders:+A+critical+review.">The developing utility of zebrafish models of neurological and neuropsychiatric disorders: A critical review.</a> Experimental Neurology. 299 (Pt A), 157-171 (2018).</li><li>Richter, K. 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=Glyoxal+as+an+alternative+fixative+to+formaldehyde+in+immunostaining+and+super-resolution+microscopy.">Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.</a> The EMBO journal. 37 (1), 139-159 (2018).</li><li>Elsalini, O. A., Rohr, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+disrupts+thyroid+function+in+developing+zebrafish.">Phenylthiourea disrupts thyroid function in developing zebrafish.</a> Development genes and evolution. 212 (12), 593-598 (2003).</li><li>Wang, W. D., Wang, Y., Wen, H. J., Buhler, D. R., Hu, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+as+a+weak+activator+of+aryl+hydrocarbon+receptor+inhibiting+2,3,7,8-tetrachlorodibenzo-p-dioxin-induced+CYP1A1+transcription+in+zebrafish+embryo.">Phenylthiourea as a weak activator of aryl hydrocarbon receptor inhibiting 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 transcription in zebrafish embryo.</a> Biochemical pharmacology. 68 (1), 63-71 (2004).</li><li>Bohnsack, B. L., Gallina, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenothiourea+sensitizes+zebrafish+cranial+neural+crest+and+extraocular+muscle+development+to+changes+in+retinoic+acid+and+IGF+signaling.">Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling.</a> PloS one. 6 (8), e22991 (2011).</li><li>Inoue, D., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+for+all--a+highly+efficient+and+versatile+method+for+fluorescent+immunostaining+in+fish+embryos.">One for all--a highly efficient and versatile method for fluorescent immunostaining in fish embryos.</a> PLoS One. 6 (5), e19713 (2011).</li></ol>

Immunohistochemistry is a versatile tool that can be used to characterize the spatio-temporal expression of virtually any protein of interest in an organism. Immunohistochemistry is used on a wide variety of tissues and model organisms. This protocol has been optimized for use in zebrafish. Immunohistochemistry in different species may require different fixation and handling techniques, blocking solutions depending on species and the presence of endogenous peroxidases, and incubation times due to the thickness and compos...

The zebrafish (Danio rerio) has emerged as a powerful model for the study of biological processes for several reasons including short generation time, rapid development, and amenability to genetic techniques. As a result, zebrafish are commonly used in high throughput small molecule screens for toxicological research and drug discovery. Zebrafish are also an attractive model for the study of developmental processes given that a single female can routinely produce 50-300 eggs at a time and the optically clear embryos develop externally allowing for efficient visualization of developmental processes. However, early research relied mostly on forward genetic screens using N-ethyl-N-nitrosourea (ENU) or other mutagens due to challenges in establishing reverse genetic techniques. Roughly two decades ago, morpholinos were first used in zebrafish to knockdown targeted genes1. Morpholinos are small antisense oligonucleotides that inhibit translation of target mRNA following microinjection into an embryo at an early developmental stage. A major weakness of morpholinos is that they are diluted as the cells divide and generally lose effectiveness by 72 hours post-fertilization (hpf). While morpholinos remain a powerful tool for zebrafish gene disruption, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPRs) are more recently being used to directly target the zebrafish genome2,3. These reverse genetic strategies, in combination with forward genetics and high throughput screens, have established the zebrafish as a powerful model to study gene expression and function.
The ability to study gene function generally requires an evaluation of the spatio-temporal distribution of gene or gene product expression. The two most commonly used techniques to visualize such expression patterns during early development are in situ hybridization (ISH) and whole mount immunohistochemistry (IHC). In situ hybridization was first developed in 1969 and relies on the use of labeled antisense RNA probes to detect mRNA expression in an organism4. In contrast, labeled antibodies are used in immunohistochemistry to visualize protein expression. The idea of labeling proteins for detection dates back to the 1930&#39;s5 and the first IHC experiment was published in 1941 when FITC-labeled antibodies were used to detect pathogenic bacteria in infected tissues6. ISH and IHC have evolved and improved significantly over the subsequent decades and are now both routinely used in the molecular and diagnostic research laboratory7,8,9,10,11. While both techniques have advantages and disadvantages, IHC offers several benefits over ISH. Practically, IHC is much less time consuming than ISH and is generally less expensive depending on the cost of the primary antibody. In addition, mRNA expression is not always a reliable metric of protein expression as it has been demonstrated in mice and humans that only about a third of protein abundance variation can be explained by mRNA abundance12. For this reason, IHC is an important supplement to confirm ISH data, when possible. Finally, IHC can provide subcellular and co-localization data that cannot be determined by ISH13,14,15. Here, we describe a step-by-step method to reliably detect proteins by immunohistochemistry in whole mount zebrafish embryos and larvae. The goal of this technique is to determine the spatial and temporal expression of a protein of interest in the whole embryo. This technology utilizes antigen-specific primary antibodies and fluorescently tagged secondary antibodies. The protocol is readily adaptable to use on slide-mounted tissue sections and for use with chromogenic substrates in lieu of fluorescence. Using this protocol, we demonstrate that developing zebrafish skeletal muscle expresses ionotropic glutamate receptors, in addition to acetylcholine receptors. NMDA-type glutamate receptor subunits are detectable on the longitudinal muscle at 23 hpf.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil, heavy duty</td>
			<td>Kirkland</td>
			<td></td>
			<td>Any brand may be substituted</td>
		</tr>
		<tr>
			<td>Anti-NMDA antibody</td>
			<td>Millipore Sigma</td>
			<td>MAB363</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H3 (Ser10), clone RR002</td>
			<td>Millipore Sigma</td>
			<td>05-598</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-pan-AMPA receptor (GluR1-4)</td>
			<td>Millipore Sigma</td>
			<td>MABN832</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate [Ca(NO3)2]</td>
			<td>Sigma Aldrich</td>
			<td>C4955</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT150C</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Sally Hanson</td>
			<td></td>
			<td>Any nail polish or hardener may be subsituted</td>
		</tr>
		<tr>
			<td>Depression (concavity) slide</td>
			<td>Electron Miscroscopy Sciences</td>
			<td>71878-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>Thermo Scientific</td>
			<td>1855920</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo medium, Danieau, 30%</td>
			<td></td>
			<td></td>
			<td>17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgS04, 0.18 mM Ca(NO3)2, 1.5 mM HEPES in ultrapure water.</td>
		</tr>
		<tr>
			<td>Embryo medium, E2</td>
			<td></td>
			<td></td>
			<td>7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO4, 75 uM KH2PO4, 25 uM Na2HPO4, 0.5 mM CaCl2, 0.35 mM NaHCO3, 0.5 mg/L methylene blue</td>
		</tr>
		<tr>
			<td>Floating tube holder</td>
			<td>Thermo Scientific</td>
			<td>59744015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence compound microscope</td>
			<td>Leica Biosystems</td>
			<td>DMi8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereomicroscope</td>
			<td>Leica Biosystems</td>
			<td>M165-FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 18 x 18</td>
			<td>Corning</td>
			<td>284518</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 22 x 60</td>
			<td>Thermo Scientific</td>
			<td>22-050-222</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Alexa 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Humid chamber with lid</td>
			<td>Simport</td>
			<td>M920-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide, 30%</td>
			<td>Fisher Scientific</td>
			<td>H325-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunedge pap pen</td>
			<td>Vector labs</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect pins, size 00</td>
			<td>Stoelting</td>
			<td>5213323</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4 &#183; 7H2O)</td>
			<td>Sigma Aldrich</td>
			<td>63138</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh strainer</td>
			<td>Oneida</td>
			<td></td>
			<td>Any brand may be substituted</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-tube cap lock</td>
			<td>Research Products International</td>
			<td>145062</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>Toastmaster</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Sigma Aldrich</td>
			<td>I8765</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Millipore Sigma</td>
			<td>S02L1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutating mixer</td>
			<td>Fisher Scientific</td>
			<td>88-861-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>04042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBTriton</td>
			<td></td>
			<td></td>
			<td>1% TritonX-100 in 1x PBS</td>
		</tr>
		<tr>
			<td>Permount mounting medium</td>
			<td>Fisher Chemical</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (glass)</td>
			<td>Pyrex</td>
			<td>3160100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (plastic)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl 2-thiourea</td>
			<td>Acros Organics</td>
			<td>207250250</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), 10x, pH 7.4</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), 1x</td>
			<td></td>
			<td></td>
			<td>1x made from 10x stock diluted in dH2O</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Fisher</td>
			<td>P250-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma Aldrich</td>
			<td>10165921001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Invitrogen</td>
			<td>AM2544</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>Spawning tank with lid and insert</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperBlock PBS</td>
			<td>Thermo Scientific</td>
			<td>37515</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost + slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue gel</td>
			<td>3M Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TNT</td>
			<td></td>
			<td></td>
			<td>100 mM Tris, pH 8.0; 150 mM NaCl; 0.1% Tween20; made in dH2O</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloracetic Acid (Cl3CCOOH)</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, ultrapure/double distilled</td>
			<td>Fisher Scientific</td>
			<td>W2-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Whole mount immunohistochemistry uses antibodies to detect the spatial pattern of protein expression in the intact animal. The basic workflow of immunohistochemistry (depicted in Figure 1) involves breeding zebrafish, raising and preparing embryos, blocking non-specific antigens, using an antigen-specific primary antibody to target the protein of interest, detecting that primary antibody with a labeled secondary antibody, mounting the specimen, and documentin...

Watch this Scientific Journal Video about Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae at JoVE.com

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

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. ...

Whole mount immunohistochemistry is a relatively quick and simple technique that allows for the spatial and temporal observation of proteins directly in a tissue or organism using labeled antibodies. The utility of this technique is that it allows for the staining of an intact tissue or an organism without having to first section it and confocal microscopy can be used to generate a three-dimensional representation of the protein expression pattern. Immunohistochemistry is widely used in biomedical research to understand the etiology and molecular mechanisms that underlie disease.It is also a valuable diagnostic tool to identify tumors in pathological samples. To begin, prepare spawning tanks filled with system water and with a mesh or slotted liner in place. Place adult zebrafish mixed sex pairs in groups in the tanks overnight.Use a 14-hour/10-hour light/dark cycle with lights coming on at 9 a.m. The next morning, with the lights on, change the spawning tank water with fresh system water to remove feces. Once eggs are laid, return the adults to home tanks.Collect the eggs by drawing them up using a transfer pipette. Transfer 50 eggs to each Petri dish filled halfway with embryo medium. Remove any opaque and cloudy eggs that are dead or failed to divide.Incubate the dish of eggs at 28.5 degrees Celsius until 23 hours post fertilization. If embryos are older than 24 hours post fertilization, with a pipette transfer the embryos to 200 micromolar PTU in embryo medium to prevent melanogenesis. Under a stereo microscope, dechorionate the unhatched embryos using ultra fine tip forceps.Using fire-polished Pasteur pipettes to minimize damage, transfer them into glass or plastic Petri dishes coated with 1-2%agarose dissolved in embryo medium. Then transfer the embryos to 1.5 milliliter centrifuge tubes using a plastic or fire-polished pipette. Remove embryo medium with a micropipette.Leave only enough liquid to just cover the embryos. Fix the embryos in 4%PFA for one to two hours with gentle rocking at room temperature. Next, wash the embryos three times in 1X PBS and 1%PBTriton for five minutes.Use the embryos immediately or store at four degrees Celsius for up to one week. For long-term storage, dehydrate and store the embryos in 100%methanol at minus 20 degrees Celsius. To rehydrate the embryos, perform serial incubations five minutes each at room temperature with decreasing amounts of methanol and PBS and PBTriton for the final wash.Proceed with the embryo preparation according to the manuscript. Select a blocking solution matching the secondary antibody host species, for example 10%goat serum in PBTriton with or without two milligrams per milliliter of BSA. Add 0.5 milliliters of the blocking solution in the tube containing the embryo and place the tube on a rocker for one to three hours at room temperature.Then incubate the embryo in primary antibody diluted in blocking solution and PBTriton overnight at four degrees Celsius while rocking. In the morning, wash the embryo five times in PBTriton for 10 minutes each at room temperature while rocking. Then select a secondary antibody based on the host species of the primary antibody and the desired wavelength at 488 nanometers.Add the secondary antibody diluted in blocking solution into the tube containing the embryo. Cover the tube with aluminum foil and incubate for two hours at room temperature while rocking. After that, wash the embryos three times in PBTriton for 10 minutes each at room temperature while covered with aluminum foil and rocking.With a pipette, transfer the embryos to a 50%glycerol solution in PBS over a bed of 2%agarose in embryo medium. To remove the yolk, transfer 200 microliters of 1X PBS to a depression slide or a plain glass slide. Use a plastic transfer pipette to move one or more embryos to the PBS droplet.Use ultra fine forceps and double zero insect pins to break apart the yolk and very gently scrape yolk granules from the ventral surface of the embryo. Remove the yolk granules and replenish with PBS as needed. After mounting, place the sample on the microscope stage.Locate the region of interest by selecting a relatively bright example. Adjust the camera exposure and gain so that the signal is sufficiently bright without saturating. When comparing between the experimental antibody-labeled embryos and control antibody embryos, use the same exposure settings.This protocol of whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression using zebrafish development. The GluN1 subunit of the NMDA type glutamate receptor revealed expression of glutamate receptor subunits throughout developing zebrafish muscles at 23 hours post fertilization. Frozen sections of 72-hour post fertilization embryos were mounted on slides and immunostained for phospho-H3, a marker of proliferating cells.Several cells expressed phospho-H3 and the expression was most notable at the ventricular zones. Mouse IgG control antibody and excluding primary antibodies revealed a low level of non-specific expression. It's important to select appropriate blocking solutions and secondary antibodies based on the primary antibodies host species to ensure detection and to minimize non-specific staining.Immunohistochemistry must be verified to ensure that the signal observed is actually the protein of interest. Followup experiments usually involve using genetically or environmentally modified organisms. Immunohistochemistry grants researchers the ability to observe proteins in situ, greatly accelerating our understanding of numerous systems in both basic and applied biology particularly during embryo development.Methanol and formaldehyde are toxic and should be handled carefully. Wastes need to be collected and disposed of according to institutional procedures.

whole-mount-immunohistochemistry-in-zebrafish-embryos-and-larvae

Research

JoVE Journal

Developmental Biology

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 membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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

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 ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

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 zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

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

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.

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.

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 ...

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 ...

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 ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

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 ...