Name: in situ hybridization combined with immunohistochemistry in cryosectioned zebrafish embryos
Uploaded: 2022-03-03T13:00:00
Description: Watch this Scientific Journal Video about In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos at JoVE.com

This work was supported by the Nantong Science and Technology Foundation of China (MS12019011), the Nantong Science and Technology Foundation of China (JC2021058), and the Natural Science Foundation of the Jiangsu Higher Education Institutions (21KJB180009).

All animal protocols were approved by the Institutional Animal Care and Use Committee of Nantong University (No. S20191210-402).
1. Collection of zebrafish embryos
<ol>
	<li>Set up a pair of zebrafish in breeding tanks the night before the eggs are to be collected, one a transgenic zebrafish and the other an AB wild-type zebrafish (Tg (foxP2:egfp-caax) X AB wild-type or Tg (hb9:egfp) X AB wild-type) (see the Table of Materials</str...

<ol>
	<li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-296 (1981).</li><li>Chen, E., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+genomics+research+model.">Zebrafish as a genomics research model.</a> Current Pharmaceutical Biotechnology. 5 (5), 409-413 (2004).</li><li>Howe, K., Clark, M. D., Torroja, C. F. <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 (7446), 498-503 (2013).</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>The lack of commercial antibodies for model organisms (and how you can deal with it) [Internet]. BenchSci Blog Available from: <a target="_blank" href="https://blog.benchsci.com/the-lack-of-commercial-antibodies-for-model-organisms-and-how-you-can-deal-with-it">https://blog.benchsci.com/the-lack-of-commercial-antibodies-for-model-organisms-and-how-you-can-deal-with-it</a> (2018)</li><li>Akam, 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=The+location+of+ultrabithorax+transcripts+in+Drosophila+tissue+sections.">The location of ultrabithorax transcripts in Drosophila tissue sections.</a> EMBO Journal. 2, 2075-2084 (1983).</li><li>Hafen, E., Levine, M., Garber, R. L., Gehring, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+in+situ+hybridization+method+for+the+detection+of+cellular+RNAs+in+Drosophila+tissue+sections+and+its+application+for+localizing+transcripts+of+the+homeotic+Antennapedia+gene+complex.">An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex.</a> EMBO Journal. 2, 617-623 (1983).</li><li>Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+on+whole-mount+zebrafish+embryos+and+young+larvae.">In situ hybridization on whole-mount zebrafish embryos and young larvae.</a> Methods in Molecular Biology. 1211, 53-67 (2014).</li><li>Clay, H., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorescent+in+situ+hybridization+in+zebrafish+embryos+using+tyramide+signal+amplification.">Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification.</a> Zebrafish. 2 (2), 105-111 (2005).</li><li>Driever, W., Stemple, D., Schier, A., Solnica-Krezel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+genetic+tools+for+studying+vertebrate+development.">Zebrafish: genetic tools for studying vertebrate development.</a> Trends in Genetics. 10 (5), 152-159 (1994).</li><li>Cunningham, R. L., Monk, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+in+situ+hybridization+and+immunohistochemistry+for+zebrafish+larvae.">Whole mount in situ hybridization and immunohistochemistry for zebrafish larvae.</a> Methods in Molecular Biology. 1739, 371-384 (2018).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature Protocols. 3 (1), 59-69 (2008).</li><li>Heisler, L. K., Zhou, L., Bajwa, P., Hsu, J., Tecott, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+5-HT(2C)+receptors+regulate+anxiety-like+behavior.">Serotonin 5-HT(2C) receptors regulate anxiety-like behavior.</a> Genes, Brain, and Behavior. 6 (5), 491-496 (2007).</li><li>Ferguson, J. L., Shive, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+immunofluorescence+and+immunohistochemistry+on+cryosectioned+zebrafish+embryos.">Sequential immunofluorescence and immunohistochemistry on cryosectioned zebrafish embryos.</a> Journal of Visualized Experiments: JoVE. (147), e59344 (2019).</li><li>Hammond-Weinberger, D. R., ZeRuth, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+immunohistochemistry+in+zebrafish+embryos+and+larvae.">Whole mount immunohistochemistry in zebrafish embryos and larvae.</a> Journal of Visualized Experiments: JoVE. (155), e60575 (2020).</li><li>Santos, D., Monteiro, S. M., Luzio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+whole-mount+immunohistochemistry+of+zebrafish+(Danio+rerio)+embryos+and+larvae+protocol.">General whole-mount immunohistochemistry of zebrafish (Danio rerio) embryos and larvae protocol.</a> Methods in Molecular Biology. 1797, 365-371 (2018).</li><li>O'Hurley, G., Sjostedt, E., Rahman, A., Li, B., Kampf, C., Ponten, F., 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=Garbage+in,+garbage+out:+a+critical+evaluation+of+strategies+used+for+validation+of+immunohistochemical+biomarkers.">Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers.</a> Molecular Oncology. 8, 783-798 (2014).</li></ol>

This protocol proposes a combination of in situ hybridization and immunohistochemistry, an important step in the colocalization experiments on zebrafish embryos. This method serves as an easy and efficient way to simultaneously analyze one mRNA and one protein. In situ hybridization and antibody staining were performed on zebrafish embryos. In contrast to several protocols published previously14,15,16, immunofl...

The zebrafish is a powerful vertebrate model for studies of development and genetics1,2. Zebrafish and humans share high genetic homology (70% of the genes are shared with the human genome), which allows its use as a model for human diseases3. In zebrafish, it is quite common to detect the expression patterns of two genes and their spatial relationship. Immunohistochemistry was first used in 1941 to detect pathogens in infected tissues by applying FITC-labeled antibodies4. The target protein in the tissue section is first labeled with a primary antibody, and the section is then labeled with a secondary antibody against the primary antibody&#39;s host species immunoglobulin. Antibody staining is a robust approach to detect the localization of proteins, which offers high optical resolution at the intracellular level. However, the number of antibodies available is very limited in zebrafish. A recent study shows that approximately 112,000 antibodies are commercially available for mice; however, very few antibodies have been demonstrated to be reliable in zebrafish5.
Instead, in zebrafish, in situ hybridization has been widely applied for gene expression pattern analysis. This method was first used to assess gene expression in Drosophila embryos in the 1980s6,7, and since then, this technology has been continuously developed and improved. Initially, radiolabeled DNA probes were used to detect mRNA transcripts; however, the spatial resolution was relatively low, and there were potential health risks caused by the radioactivity. Subsequently, in situ hybridization relies on the RNA probes labeled with digoxigenin (DIG) or fluorescein (Fluo), which are conjugated to alkaline phosphatase (AP) or detected by fluorescent tyramide signal amplification (TSA)8,9. Although TSA has been used to detect two or three genes, DIG labeling of RNA probes and antiDIG AP-conjugated antibody are still highly sensitive, stable, and widely used approaches for in situ hybridization. Therefore, commercialized antibodies combined with DIG-labeled in situ probes are useful for providing insight into protein localization and expression of one gene.
Whole-mount embryos cannot reveal the spatial relationship between genes due to the low optical resolution, even though zebrafish embryos are small and transparent10. Hence, sectioning is necessary to analyze the expression patterns of genes at the intracellular level. Cryosectioning has been widely used in zebrafish as it is easy to perform and can effectively preserve the antigen. Therefore, in situ hybridization combined with immunohistochemistry in zebrafish cryosections offers a powerful way for analyzing the expression patterns of two genes. A combination of in situ hybridization and immunohistochemistry has been applied to zebrafish11. However, proteinase K treatment was used to enhance probe penetration at the expense of antigen integrity. To overcome this limitation, this protocol uses heating to induce antigen retrieval. This protocol is not only applicable to embryos of different stages and tissue sections of various thicknesses (14 &#181;m head sections and 20 &#181;m spinal cord sections), but it has also been verified by using genes expressed in two organs, including the head and spinal cord.
This article will describe how to combine in situ hybridization and antibody staining in zebrafish embryos in cryosections. The versatility of this protocol is demonstrated by using a number of in situ hybridization-immunohistochemistry combinations, including in situ hybridization probes for two different neurons. This method is suitable for detecting mRNA and protein in different regions and embryos of different ages, as well as the expression patterns of two genes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin AP Fab fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Millipore</td>
			<td>MAB3580</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.1% Triton X-100, 3% BSA, 10% goat serum in 1x PBS</td>
		</tr>
		<tr>
			<td>BM purple</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>B2064</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate buffer</td>
			<td>Leagene</td>
			<td>IH0305</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma</td>
			<td>C2404</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold for tissue, 15 mm x 15 mm x 5 mm</td>
			<td>Head Biotechnology</td>
			<td>H4566</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC-Treated Water</td>
			<td>Sangon Biotech</td>
			<td>B501005</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera, fluorescence microscope</td>
			<td>Nikon</td>
			<td>NI-SSR 931479</td>
			<td></td>
		</tr>
		<tr>
			<td>E3 embryo medium</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>AM9342</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>J&#38;K Scientific</td>
			<td>542858</td>
			<td></td>
		</tr>
		<tr>
			<td>HYB</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>preHYB plus 50 &#181;g/mL heparin sodium salt, 100 &#181;g/mL ribonucleic acid diethylaminoethanol salt</td>
		</tr>
		<tr>
			<td>Immunohistochemical wet box</td>
			<td>Mkbio</td>
			<td>MH10002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Low profile leica blades</td>
			<td>Leica</td>
			<td>819</td>
			<td></td>
		</tr>
		<tr>
			<td>MABT (1x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.1 M maleic acid, 0.15 M NaCl, 0.02% Tween-20, pH 7.5</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J&#38;K Scientific</td>
			<td>116481</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Macklin</td>
			<td>M859248</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NTMT</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.1M Tris-HCl, 0.1M NaCl, 1% Tween-20</td>
		</tr>
		<tr>
			<td>OCT medium</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Enzo Life Sciences</td>
			<td>ADI-950-233</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 4%</td>
			<td>Abbexa</td>
			<td>abx082483</td>
			<td>made in lab in 1x PBS</td>
		</tr>
		<tr>
			<td>PBST (1x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>1x PBS plus 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Phenylthiourea</td>
			<td>Merck</td>
			<td>103-85-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (10x)</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>preHYB</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>50% formamide, 5x SSC, 9.2 mM citric acid (pH 6.0), 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>1092766</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleic acid diethylaminoethanol salt</td>
			<td>Sigma</td>
			<td>R3629</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free 1.5 mL tubes</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC (20x)</td>
			<td>Invitrogen</td>
			<td>AM9770</td>
			<td></td>
		</tr>
		<tr>
			<td>SSCT (0.2x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.2x SSC plus 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>SSCT (1x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>1x SSC plus 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>Laboratory Animal Center of Nantong University</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ hybridization combined with immunohistochemistry in cryosectioned zebrafish embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

This protocol can be used to simultaneously examine the expression pattern of one mRNA and one protein. Figure 1 shows the experimental workflow. The 5-HT2C receptor is a subtype of the 5-HT receptor bound by the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). It is widely distributed in the central nervous system (CNS) and can significantly regulate a variety of brain functions, including appetite, mood, anxiety, and reproductive behavior13. The expression of...

Watch this Scientific Journal Video about In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos at JoVE.com

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

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