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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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, immunofluorescence and immunohistochemistry were used to explore protein expression. Few zebrafish-specific antibodies have been appropriately validated for use in sections17. Previous studies have used only one technique: immunofluorescence, immunohistochemistry, or in situ hybridization. Combining in situ hybridization and immunohistochemistry to analyze RNA and protein overcomes the limitations of any one technique. The first step was in situ hybridization, which greatly protects mRNA from degradation. Antigen retrieval was induced by heating to avoid the disruption by proteinase K treatment of antibody binding during immunohistochemical staining. Whether this approach works for all antigens will require further testing. Images of the sections showed that the 5-HT2C receptor was coexpressed in foxP2 neurons and that the insm1a receptor was coexpressed in hb9 neurons.
Whole-mount in situ hybridization greatly preserves the integrity of samples. In situ hybridization followed by sectioning can decrease the incubation time for antibody staining. In addition, antibody staining after in situ hybridization can help avoid fluorescence quenching.
The following specific steps are essential to the success of the experiment. The first critical step is to refix the embryo in 4% PFA. This step stops the proteinase K reaction because PFA inactivates proteinase K. The samples should be mixed gently to expose all embryos to PFA; the tube can also be placed on its side so that the embryos are evenly distributed in the solution. The second critical step is the treatment of the embryo during OCT implantation. The embryos should be arranged in a specific direction (either dorsal-ventral or lateral), keeping them straight so that they can be cut into sections from roughly the same area for all embryos. A small needle is used to make small, conscious movements in the viscous OCT medium to locate the embryo and remove bubbles.
There are several potential modifications that can be applied to the described scenario. The digestion time of proteinase K in in situ hybridization can be determined according to the different developmental stages of the zebrafish embryos (Table 1). In addition, the thickness of the cryosections is very flexible and can be determined according to experimental requirements. While it is predicted that this approach will be suitable for a wide range of experiments, it has some potential limitations. The successful execution of this procedure depends on maintaining a complete sample through two successive experiments. There are multiple possible pause points in this protocol. The dehydrated embryos can be stored in 30% sucrose solution at -20 &#176;C for several months14. Similarly, the prepared slides can be stored in a slide box at -20 &#176;C for more than 1 year14. The frozen blocks can be stored at -80 &#176;C for three months.
This protocol for in situ hybridization combined with immunohistochemistry has been shown to be successful in detecting 5-HT2C and insm1a expression in zebrafish embryos and can be easily applied to a variety of tissues at different developmental stages. In addition, this protocol can be applied to neurons or glial cells, providing a robust investigational approach for neuroscientists.

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><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 reagent</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>Blocking solution-1</td><td>made in lab</td><td>N/A</td><td>Dissolve the blocking reagent in 1X MAB to a final concentration of 10% (wt/vol). Autoclave and store at -20 &amp;#176;C before use.</td></tr><tr><td>Blocking solution-2</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</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&amp;amp;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 &amp;micro;g/mL heparin sodium salt, 100 &amp;micro;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&amp;amp;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>MgSO&lt;sub&gt;4&lt;/sub&gt;</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 5-HT2C in the CNS was detected by the transgenic line Tg (foxp2:egfp-caax), in which foxP2 neurons are labeled by fluorescein green fluorescent protein (GFP). GFP was not expressed in wild-type zebrafish but only in the transgenic line. Simultaneous detection of the expression of RNA (5-HT2C, the probe for htr2c, Figure 2A) and protein (GFP, antiGFP, Figure 2B) can be used for protein and mRNA colocalization analysis.
Insulinoma-associated 1a (insm1a), a zinc-finger transcription factor, was first identified from the tumor reduction library and played several functions in the formation and differentiation of the vertebrate central and peripheral nervous system and neuroendocrine system. Recent studies have shown that insm1a is an important regulator of motor neuron development. The expression of insm1a in the zebrafish spinal cord and motor neurons was detected by the transgenic line Tg (hb9:egfp), in which hb9 neurons are labeled by fluorescein GFP. GFP was not expressed in wild-type zebrafish but only in the transgenic line. The simultaneous detection of the expression of RNA (insm1a, the probe for insm1a, Figure 3A) and protein (GFP, antiGFP, Figure 3B) can be used for protein and mRNA colocalization analysis. This protocol was successfully applied to detect the colocalization of protein and RNA to understand their spatial relationship.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63715/63715fig01.jpg" /> 
Figure 1: Outline of the method. This flowchart shows an experimental workflow. The workflow can be completed in a minimum of 9 days (see the number of days in the upper right corner of each phase in the workflow), although some steps can be completed in a longer period, as described in the protocol. Abbreviations: PFA = paraformaldehyde; O/N = overnight; RT = room temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63715/63715fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63715/63715fig02.jpg" /> 
Figure 2: Whole-mount embryos stained with 5-HT2C probe and GFP antibody in Tg (foxP2:egfp-caax). Bright-field (A), fluorescent (B), and merged images (C) show 5-HT2C (the probe for htr2c, A) and GFP (antiGFP, B) expression in foxP2 neurons and axon tracts. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63715/63715fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63715/63715fig03.jpg" /> 
Figure 3: Whole-mount embryos stained with insm1a probe and GFP antibody in Tg (hb9:egfp). Bright-field (A), fluorescent (B), and merged images (C) show insm1a (the probe for insm1a, A) and GFP (anti-GFP, B) expression in hb9 neurons. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63715/63715fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Embryos</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>24 hpf</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>30 hpf</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>36 hpf</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>48 hpf</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>72 hpf</td>
			<td>15 min</td>
		</tr>
		<tr>
			<td>96 hpf</td>
			<td>45 min</td>
		</tr>
		<tr>
			<td>4-5 dpf</td>
			<td>60 min</td>
		</tr>
	</tbody>
</table>
Table 1: Proteinase K digestion time for zebrafish embryos. Abbreviations: hpf = hours post fertilization; dpf = days post fertilization.

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.7K 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

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Chemistry

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 ...

Developmental Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biology

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

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

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

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

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

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

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

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

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

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

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

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

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

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

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Chapters in this video

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Immunostaining of Dissected Zebrafish Embryonic Heart

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization