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). Use a diagonal plastic divider to separate the male and female to preclude physical access. The following day, fit the upper part of the breeding tank into a clean lower part filled with fresh water, and remove the divider of the breeding tank. Allow the fish mate for 10-20 min, and collect the eggs after they have sunk to the bottom of the breeding tank.</li>
	<li>Culture the embryos in E3 embryo medium (see the Table of Materials) containing methylene blue (1 mL of 0.05% methylene blue in 1 L of E3 embryo medium) at 28.5 &#176;C.</li>
	<li>Treat the embryos at 24 h post fertilization (hpf) with phenylthiourea (PTU, see the Table of Materials) to prevent pigment formation. 
	&#8203;NOTE: Animals of either sex are used in experiments.</li>
</ol>
2. In situ hybridization
NOTE: The water used for steps 2.1-2.11 is diethyl pyrocarbonate (DEPC)-treated water (see the Table of Materials).
<ol>
	<li>Fix ~10 embryos with 0.5-1 mL of fresh 4% paraformaldehyde (PFA, see the Table of Materials) in a 1.5 mL microfuge tube at 4 &#176;C overnight for 12-14 h. 
	NOTE: The protocol for in situ hybridization is modified slightly from previously published literature12. PFA must be freshly prepared and stored at 4 &#176;C within one week of use or for one month at -20 &#176;C. All the following steps in in situ hybridization were performed using 1.5 mL microfuge tubes.</li>
	<li>Use tweezers to remove the skin of only embryos older than 48 hpf. 
	NOTE: The skin is removed to facilitate the penetration of RNA probes into the trunk region of embryos older than 48 hpf.&#160;</li>
	<li>Gradually dehydrate the embryos by washing with 25%, 50%, and 75% methanol in 1x phosphate-buffered saline containing 0.1% Tween-20 successively (PBST, see the Table of Materials) for 5 min each at room temperature. Then, wash the embryos for 5 min in 100% methanol at room temperature. Incubate the embryos in 100% methanol at -20 &#176;C for at least overnight for 12-14 h. 
	NOTE: Dehydrated embryos can be stored in 100% methanol at -20 &#176;C for 6 months.</li>
	<li>Gradually rehydrate the embryos by washing with 75%, 50%, and 25% methanol in PBST successively for 5 min each at room temperature. Wash the embryo three times with PBST for 5 min each at room temperature.</li>
	<li>Digest the embryos with 10 &#181;g/mL proteinase K (see the Table of Materials) in PBST at room temperature (see Table 1).</li>
	<li>Wash the embryos with PBST for 5 min. Perform this washing step three times.</li>
	<li>Refix the washed embryos in 4% PFA for 15 min at room temperature. 
	NOTE: This step stops the digestion because PFA inactivates proteinase K. Ensure that the sample is mixed gently to expose all the embryos to PFA; the tubes can be placed on their sides to evenly distribute the embryos in the solution. This PFA does not have to be fresh (it can be refrigerated for up to 2 weeks).</li>
	<li>Wash the embryo three times with PBST, incubating for 5 min during each wash.</li>
	<li>Perform prehybridization of the embryos by incubating with prehybridization solution (preHYB, see the Table of Materials) at 65 &#176;C for 5 min. Replace preHYB with hybridization solution (HYB, see the Table of Materials) and prehybridize for at least 4 h in HYB. 
	NOTE: Before proceeding with prehybridization, preheat the solutions to 65 &#176;C. Formamide (see the Table of Materials) is used to maintain the shape and structure of the tissue. Formamide also prevents the binding of nonhomologous fragments at low temperatures.</li>
	<li>Heat the probe (Insm1a or 5-HT2C) in HYB for 5 min at 95 &#176;C before adding to the embryos. Use the probe at 1 &#181;g/mL HYB. Remove as much preHYB as possible without letting the embryos come into contact with the air, and add preheated probe in HYB to the tube containing the embryos. 
	NOTE: A labeled RNA probe can be used to hybridize with a target mRNA sequence in the embryos. Therefore, the probe can be used to detect the expression of a gene of interest and the location of mRNA.</li>
	<li>Allow the probe to hybridize overnight (12-14 h) at 50-70 &#176;C. 
	NOTE: The hybridization temperature differs for different probes.</li>
	<li>The next day, aspirate the probe solution with a pipette and store it in a tube at -20 &#176;C so that it can be reused many times.</li>
	<li>Wash the embryos as follows:
	<ol>
		<li>Wash the embryos for 15 min with 100% HYB at 65 &#176;C.</li>
		<li>Wash the embryos sequentially with 75%, 50%, and 25% HYB in 2x standard saline citrate containing 0.1% Tween-20 (SSCT, see the Table of Materials) for 15 min each at 65 &#176;C.</li>
		<li>Wash the embryos for 15 min in 2x SSCT at 65 &#176;C.</li>
		<li>Wash the embryos for 15 min in 0.2x SSCT at 65 &#176;C.</li>
	</ol>
	</li>
	<li>Wash the embryos two times for 10 min in maleic acid buffer containing 0.02% Tween-20 (MABT, see the Table of Materials) at room temperature.</li>
	<li>Block the hybridized and washed embryos for at least 2 h at room temperature with 2% blocking solution-1 (see the Table of Materials).</li>
	<li>Replace the blocking solution-1 with antidigoxigenin AP (1:4,000 dilution, see the Table of Materials) in a fresh 2% blocking solution-1 and shake overnight for 12-14 h at 4 &#176;C.</li>
	<li>Wash the embryos four times for 30 min in MABT at room temperature. 
	NOTE: Remove the BM purple (see the Table of Materials) from the refrigerator during the third wash and shake it occasionally during the subsequent washes.</li>
	<li>Wash the embryos two times for 10 min in NTMT (0.1 M Tris-HCl, 0.1 M NaCl, 1% Tween-20, see the Table of Materials).</li>
	<li>Use a pipette to remove as much NTMT as possible from the embryos. Replace with BM purple AP substrate and stain the embryos at room temperature in the dark. Monitor the color changes every 30 min to control the degree of dyeing. 
	NOTE: The specific dyeing time is different and needs to be adjusted according to each probe. Incubating the embryos at 37 &#176;C can accelerate the reaction. Incubating the embryos at 4 &#176;C can increase the reaction time and can be done overnight.</li>
	<li>Once it is developed to the desired extent, stop the reaction by briefly rinsing with NTMT two times. After in situ hybridization, rinse the embryos with PBST thrice for 20 min.</li>
</ol>
3. Embedding
<ol>
	<li>Immerse the embryos in 5% sucrose in 1x PBS (see the Table of Materials) overnight for 12-14 h at 4 &#176;C.</li>
	<li>Change the solution covering the embryos to 15% sucrose in 1x PBS and incubate overnight for 12-16 h at 4 &#176;C.</li>
	<li>Change the solution covering the embryos to 30% sucrose in 1x PBS and incubate for 1-2 days at 4 &#176;C. 
	NOTE: Incubate in this solution until the embryos sink to the bottom of the tube.</li>
	<li>Fill a cryomold with optimal cutting temperature (OCT) medium (see the Table of Materials). Transfer the embryos in 30% sucrose to the cryomold with OCT medium. Stir them to remove the sucrose from the embryos.</li>
	<li>Transfer the embryos to a new cryomold for tissue and gently fill it with OCT medium, avoiding the formation of bubbles.</li>
	<li>Submerge each embryo, pushing it to the bottom of the cryomold, and place each embryo in the desired orientation (either dorsal-ventral or lateral). Keep the embryos in a straight line. 
	NOTE: It is strongly recommended to place only one embryo in each cryomold (see the Table of Materials).</li>
	<li>Place the embedded embryos in cryomolds in a dry ice ethanol bath.</li>
	<li>Store at -80 &#176;C at least overnight for 12-14 h. 
	&#8203;NOTE: The cryomolds can be kept at -80 &#176;C for at least one month.</li>
</ol>
4. Cryosectioning
<ol>
	<li>Set the cryosections using a cryostat to -20 &#176;C.</li>
	<li>Remove the specimen block from the cryomold and place it in the cryostat. Place OCT medium on the base of the chilled chuck and place the block on top.</li>
	<li>Ensure that the specimen block is parallel to the razor blade. Carefully trim off excess OCT medium around the specimen.</li>
	<li>Cut into 12-20 &#181;m thick sections using a cryostat. Quickly transfer the sections to glass slides so that each slide has 3-4 sections. Allow the samples to reach room temperature, and store the sections in a sealed slide box at -80 &#176;C for later use.</li>
</ol>
5. Immunostaining
NOTE: GFP staining is performed on the sections.
<ol>
	<li>Wash the slides containing the sections with PBS for 5 min.</li>
	<li>Heat citrate buffer to boiling in a microwave.</li>
	<li>Place the slides in the buffer and continue to heat to keep the solution near boiling for approximately 20 min. 
	NOTE: This step helps in antigen retrieval. The tissue remains intact even at high temperatures, which improves staining quality by preventing folding, damage, or detachment of the tissues.</li>
	<li>Let the samples cool slowly to room temperature prior to the next step. Drain the excess solution, carefully dry the area around each section with a piece of tissue, and draw a circle around the section with a water-repellant pen (see the Table of Materials) to form a hydrophobic barrier. Be careful not to dry the tissue sections.</li>
	<li>Wash the slides two times with PBS, incubating for 10 min during each wash.</li>
	<li>Block for 2 h in blocking solution-2 (see the Table of Materials) at room temperature.</li>
	<li>Pipette primary antibody solution (mouse monoclonal &#945;-GFP, 1:250, see the Table of Materials) per slide and incubate the slides in an immunohistochemical wet box at 4 &#176;C overnight.</li>
	<li>Wash the slides three times with PBS, incubating for 10 min during each wash.</li>
	<li>Drain excess PBS. Incubate the slides with the appropriate secondary antibody (1:400, see the Table of Materials) for 1 h at room temperature in PBS.</li>
	<li>Wash the slides three times with PBS, incubating for 10 min during each wash.</li>
	<li>Drain the excess PBS, pipette the mounting medium onto the slide, and mount with a slide coverslip.</li>
</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=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]. 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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. 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The authors have no conflicts of interest to disclose.

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 ...

in-situ-hybridization-combined-with-immunohistochemistry

Research

JoVE Journal

Neuroscience

2.6K Views.  Nantong University. 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.

Video: In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...