Summary
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.
Abstract
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.
Introduction
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'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 µm head sections and 20 µm 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.
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Protocol
All animal protocols were approved by the Institutional Animal Care and Use Committee of Nantong University (No. S20191210-402).
1. Collection of zebrafish embryos
- 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.
- 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 °C.
- Treat the embryos at 24 h post fertilization (hpf) with phenylthiourea (PTU, see the Table of Materials) to prevent pigment formation.
NOTE: Animals of either sex are used in experiments.
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).
- 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 °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 °C within one week of use or for one month at -20 °C. All the following steps in in situ hybridization were performed using 1.5 mL microfuge tubes. - 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. - 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 °C for at least overnight for 12-14 h.
NOTE: Dehydrated embryos can be stored in 100% methanol at -20 °C for 6 months. - 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.
- Digest the embryos with 10 µg/mL proteinase K (see the Table of Materials) in PBST at room temperature (see Table 1).
- Wash the embryos with PBST for 5 min. Perform this washing step three times.
- 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). - Wash the embryo three times with PBST, incubating for 5 min during each wash.
- Perform prehybridization of the embryos by incubating with prehybridization solution (preHYB, see the Table of Materials) at 65 °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 °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. - Heat the probe (Insm1a or 5-HT2C) in HYB for 5 min at 95 °C before adding to the embryos. Use the probe at 1 µg/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. - Allow the probe to hybridize overnight (12-14 h) at 50-70 °C.
NOTE: The hybridization temperature differs for different probes. - The next day, aspirate the probe solution with a pipette and store it in a tube at -20 °C so that it can be reused many times.
- Wash the embryos as follows:
- Wash the embryos for 15 min with 100% HYB at 65 °C.
- 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 °C.
- Wash the embryos for 15 min in 2x SSCT at 65 °C.
- Wash the embryos for 15 min in 0.2x SSCT at 65 °C.
- 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.
- Block the hybridized and washed embryos for at least 2 h at room temperature with 2% blocking solution-1 (see the Table of Materials).
- 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 °C.
- 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. - 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).
- 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 °C can accelerate the reaction. Incubating the embryos at 4 °C can increase the reaction time and can be done overnight. - 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.
3. Embedding
- Immerse the embryos in 5% sucrose in 1x PBS (see the Table of Materials) overnight for 12-14 h at 4 °C.
- Change the solution covering the embryos to 15% sucrose in 1x PBS and incubate overnight for 12-16 h at 4 °C.
- Change the solution covering the embryos to 30% sucrose in 1x PBS and incubate for 1-2 days at 4 °C.
NOTE: Incubate in this solution until the embryos sink to the bottom of the tube. - 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.
- Transfer the embryos to a new cryomold for tissue and gently fill it with OCT medium, avoiding the formation of bubbles.
- 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). - Place the embedded embryos in cryomolds in a dry ice ethanol bath.
- Store at -80 °C at least overnight for 12-14 h.
NOTE: The cryomolds can be kept at -80 °C for at least one month.
4. Cryosectioning
- Set the cryosections using a cryostat to -20 °C.
- 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.
- Ensure that the specimen block is parallel to the razor blade. Carefully trim off excess OCT medium around the specimen.
- Cut into 12-20 µm 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 °C for later use.
5. Immunostaining
NOTE: GFP staining is performed on the sections.
- Wash the slides containing the sections with PBS for 5 min.
- Heat citrate buffer to boiling in a microwave.
- 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. - 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.
- Wash the slides two times with PBS, incubating for 10 min during each wash.
- Block for 2 h in blocking solution-2 (see the Table of Materials) at room temperature.
- Pipette primary antibody solution (mouse monoclonal α-GFP, 1:250, see the Table of Materials) per slide and incubate the slides in an immunohistochemical wet box at 4 °C overnight.
- Wash the slides three times with PBS, incubating for 10 min during each wash.
- 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.
- Wash the slides three times with PBS, incubating for 10 min during each wash.
- Drain the excess PBS, pipette the mounting medium onto the slide, and mount with a slide coverslip.
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Representative Results
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.
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. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
| Embryos | Time |
| 24 hpf | 2 min |
| 30 hpf | 3 min |
| 36 hpf | 5 min |
| 48 hpf | 10 min |
| 72 hpf | 15 min |
| 96 hpf | 45 min |
| 4-5 dpf | 60 min |
Table 1: Proteinase K digestion time for zebrafish embryos. Abbreviations: hpf = hours post fertilization; dpf = days post fertilization.
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Discussion
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 °C for several months14. Similarly, the prepared slides can be stored in a slide box at -20 °C for more than 1 year14. The frozen blocks can be stored at -80 °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.
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Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
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).
Materials
| Name | Company | Catalog Number | Comments |
| Alexa Fluor 488 secondary antibody | Invitrogen | A21202 | |
| Anti-Digoxigenin AP Fab fragments | Roche | 11093274910 | |
| Anti-GFP antibody | Millipore | MAB3580 | |
| Blocking reagent | Roche | 11096176001 | |
| Blocking solution-1 | made in lab | N/A | Dissolve the blocking reagent in 1X MAB to a final concentration of 10% (wt/vol). Autoclave and store at -20 °C before use. |
| Blocking solution-2 | made in lab | N/A | 0.1% Triton X-100, 3% BSA, 10% goat serum in 1x PBS |
| BM purple | Roche | 11442074001 | |
| Bovine Serum Albumin (BSA) | Sigma | B2064 | |
| CaCl2 | Sigma | C5670 | |
| Citrate buffer | Leagene | IH0305 | |
| Citric acid | Sigma | C2404 | |
| Cryomold for tissue, 15 mm x 15 mm x 5 mm | Head Biotechnology | H4566 | |
| DEPC-Treated Water | Sangon Biotech | B501005 | |
| Digital camera, fluorescence microscope | Nikon | NI-SSR 931479 | |
| E3 embryo medium | made in lab | N/A | 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 |
| Formamide | Invitrogen | AM9342 | |
| Goat serum | Sigma | G9023 | |
| Heparin sodium salt | J&K Scientific | 542858 | |
| HYB | made in lab | N/A | preHYB plus 50 µg/mL heparin sodium salt, 100 µg/mL ribonucleic acid diethylaminoethanol salt |
| Immunohistochemical wet box | Mkbio | MH10002 | |
| KCl | Sigma | P5405 | |
| Low profile leica blades | Leica | 819 | |
| MABT (1x) | made in lab | N/A | 0.1 M maleic acid, 0.15 M NaCl, 0.02% Tween-20, pH 7.5 |
| Maleic acid | Sigma | M0375 | |
| Methanol | J&K Scientific | 116481 | |
| Methylene blue | Macklin | M859248 | |
| MgSO4 | Sigma | M2643 | |
| NaCl | Sigma | S5886 | |
| NTMT | made in lab | N/A | 0.1M Tris-HCl, 0.1M NaCl, 1% Tween-20 |
| OCT medium | Tissue-Tek | 4583 | |
| PAP pen | Enzo Life Sciences | ADI-950-233 | |
| Paraformaldehyde, 4% | Abbexa | abx082483 | made in lab in 1x PBS |
| PBST (1x) | made in lab | N/A | 1x PBS plus 0.1% Tween-20 |
| Phenylthiourea | Merck | 103-85-5 | |
| Phosphate-buffered saline (10x) | Invitrogen | AM9624 | |
| preHYB | made in lab | N/A | 50% formamide, 5x SSC, 9.2 mM citric acid (pH 6.0), 0.1% Tween-20 |
| Proteinase K | Roche | 1092766 | |
| Ribonucleic acid diethylaminoethanol salt | Sigma | R3629 | |
| RNase-free 1.5 mL tubes | Ambion | AM12400 | |
| SSC (20x) | Invitrogen | AM9770 | |
| SSCT (0.2x) | made in lab | N/A | 0.2x SSC plus 0.1% Tween-20 |
| SSCT (1x) | made in lab | N/A | 1x SSC plus 0.1% Tween-20 |
| Sucrose | Invitrogen | 15503022 | |
| Triton X-100 | Sigma | T9284 | |
| Tween-20 | Sigma | P1379 | |
| Zebrafish | Laboratory Animal Center of Nantong University | N/A |
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