Name: using immunofluorescence to detect pm2.5-induced dna damage in zebrafish embryo hearts
Uploaded: 2021-02-15T13:00:00.000+00:00
Duration: 5 min 58 s
Description: Watch this Scientific Journal Video about Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts at JoVE.com

This work was supported by the National Nature Sciences Foundation of China (Grant number: 81870239, 81741005, 81972999) and The Priority Academic Program Development of Jiangsu Higher Education Institutions.

Wild type zebrafish (AB) used in this study were obtained from the National Zebrafish Resource Center in Wuhan, China. All animal procedures outlined here have been reviewed and approved by the Animal Care Institution of The Ethics Committee of Soochow University.
1. PM2.5 sampling and organic compound extraction
NOTE: PM2.5 was collected in an urban area in Suzhou, China, August 1-7, 2015, as described previously5.
<ol>
	<li>Bake 47 mm quartz membrane filters in a 500 &#176;C muffle furnace for 2 h to remove the organic components.</li>
	<li>Place a filter in a PM2.5 sampler for 24 h of uninterrupted sampling.</li>
	<li>Remove the filter and dry for 24 h at room temperature.</li>
	<li>Quantify the filter with an analytical balance.</li>
	<li>Extract organic components from the filter by Soxhlet extraction using dichloromethane as a solvent19.</li>
	<li>Dry the EOM by a rotary evaporation in a 60 &#176;C water bath and nitrogen flow. Dissolve EOM in DMSO and store at -20 &#176;C.</li>
</ol>
2. Zebrafish embryo collection and treatment
<ol>
	<li>Maintain the zebrafish at 28.5 &#177; 0.5 &#176;C in a re-circulating aquaculture system with a 14 h light and 10 h dark photoperiod cycle.</li>
	<li>Place healthy adult zebrafish into a tank at a 2:1 male to female ratio.</li>
	<li>The next day, collect the embryos and wash them with system water (i.e., zebrafish breeding water).</li>
	<li>Select and randomly divide zebrafish embryos demonstrating a normal development (uniform size, full grains, and no egg coagulation) into 4 groups in individual glass Petri dishes with a diameter of 7 cm (about 50 embryos per dish).</li>
	<li>Treat the embryos with PM2.5 (5 mg/L) in the presence or absence of NAC at 0.25 &#956;M from 2 hpf until 72 hpf. Use DMSO as a vehicle control to a final concentration at 0.1% (v/v).</li>
</ol>
3. Morphological observation of zebrafish embryos and cardiac dissection
<ol>
	<li>At 72 hpf, transfer embryos to glass slides and observe under a stereo microscope. Record heart malformations, such as pericardial edema, altered looping, and decreased size.</li>
	<li>Calculate malformation rates (the percentage of embryos with heart defects out of the total living embryos) and analyze differences between groups using one-way ANOVA followed by Turkey&#39;s Multiple Comparison Test (p &#60; 0.05 = statistically significant).</li>
	<li>Anesthetize the embryos with 0.6 mg/mL MS-222 to immobilize them on glass slides.</li>
	<li>Record heart beats for 30 s and quantify heart rates using ImageJ software5,20.</li>
	<li>Dissect hearts from zebrafish embryos with a disposable syringe needle under a stereo microscope. Caution: Avoid destroying the heart shape.</li>
</ol>
4. Immunofluorescence Assay
<ol>
	<li>To use an immunofluorescence assay to detect PM2.5 induced DNA damage in the heart of zebrafish embryos, use a hydrophobic barrier pen to draw a circle on a clean glass side.</li>
	<li>Add 50 &#956;L of 4% paraformaldehyde to 1.25 mL of Phosphate Buffered Saline (PBS) to make a fixative solution.</li>
	<li>Place 3 dissected hearts into one hydrophobic barrier pen circle and incubate for 20 min at room temperature.</li>
	<li>Decant the solution under the microscope and dry the samples at room temperature for at least 5 min, so that the hearts completely attach to the glass slides.</li>
	<li>Wash the slides three times in PBS with 0.1% Tween 20 (PBST) for 5 min per wash.</li>
	<li>Add 50 &#956;L of bovine serum albumin (BSA) to 1000 &#956;L of PBST to obtain a 5% BSA solution and incubate the slides in a humid chamber for 1 h to block non-specific antibody binding.</li>
	<li>Decant the solution and wash the samples three times with PBST for 5 min per wash.</li>
	<li>Dilute 2 &#956;L of mouse monoclonal antibody against 8-OHdG and 2 &#956;L of rabbit polyclonal antibody against &#947;H2AX in 296 &#956;L of PBST to obtain a working primary antibody cocktail solution.</li>
	<li>Incubate the heart samples with 50 &#956;L of the primary antibody cocktail solution against 8-OHdG and &#947;H2AX in a humidified chamber for at least one hour at room temperature or overnight at 4 &#176;C (Overnight incubation can increase signal intensity).</li>
	<li>Decant the solution and wash the samples three times with PBST for 5 min per wash.</li>
	<li>Dilute 1 &#956;L of FITC-labeled goat anti-mouse secondary antibody and 1 &#956;L of cy3 goat anti-rabbit secondary antibody in 498 &#956;L of PBST to obtain a working secondary antibody cocktail solution and incubate the samples with the secondary antibodies (1:500 in PBST) for 1 h at room temperature in the dark.</li>
	<li>Decant the solution and wash the samples three times with PBS for 5 min per wash protected from light.</li>
	<li>Add 20 &#956;L of DAPI (4&#39;,6-diamidino-2-phenylindole) to the samples for nuclear staining for 30 min at room temperature.</li>
	<li>Apply a coverslip to slide and seal with nail polish to prevent drying and movement. Then image the samples under a fluorescence microscope and quantify the fluorescence signal of heart area using ImageJ software. Calculate the relative changes with the average of DMSO control samples. Determine the statistical significance of the data as in step 3.2.</li>
</ol>

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The authors have nothing to disclose.

Although zebrafish is an excellent vertebrate model for studying the cardiac developmental toxicity of environmental chemicals, due to the small size of the embryo heart, it is difficult to obtain enough protein for western blot analysis. Therefore, we present a sensitive immunofluorescence method for quantifying the protein expression levels of DNA damage biomarkers in the hearts of zebrafish embryos exposed to PM2.5.
During dissection, it is important to keep the integrity of the heart intact. In our experience, it is relatively easy to perform the isolation at 72 hpf. In addition, the heart needs to be put into fixation solution as soon as possible after collection. Another critical step is drying the samples to make sure that the dissected hearts are completely attached to the glass slide. Otherwise, the samples may be washed off from the slide during labeling.
Dual immunofluorescence staining is performed to detect both 8-OHdG and &#947;H2AX signals in the isolated hearts. This method not only saves labor and allows the use of a reduced sample size, but also facilitates co-localization of the two signals. Although this antibody-based method can not detect fluorescence signals in living embryos, this rapid protocol can be used to detect protein expression in isolated zebrafish embryo hearts.
It has been frequently reported that oxidative stress mediates PM2.5-induced DNA damage8,9. Excessive ROS production can lead to DNA damage and apoptosis during zebrafish embryonic development21,22,23. As we have previously reported18, an increased 8-OHdG and &#947;H2AX signal expression is observed in the hearts of zebrafish embryos exposed to EOM, the expressions of which are significantly counteracted by treatment with the ROS scavenger NAC. It is noteworthy that NAC does not completely reverse PM2.5-induced DNA damage signal expression, indicating that oxidative stress may only contribute partially to the DNA damage observed in the hearts of zebrafish embryos exposed to PM2.5.
In conclusion, this method uses a sensitive technique for detecting PM2.5 induced DNA damage in the intact hearts of zebrafish embryos. In addition, the method can be applied to detect protein expression changes in the hearts of zebrafish embryos exposed to other environmental chemicals.

Air pollution is now a serious environmental problem facing the world. Ambient fine particulate matter (PM2.5), which is one of the most important indicators of air quality, can carry a large number of harmful substances and enter the blood circulatory system, causing serious harm to human health1. Epidemiology studies have demonstrated that PM2.5 exposure can lead to an increased risk of congenital heart defects (CHDs)2,3. Evidence from animal experiments also showed that PM2.5 can cause abnormal cardiac development in zebrafish embryos and the offspring of mice but the molecular mechanisms of the cardiac developmental toxicity of PM2.5 is still largely unknown4,5,6.
DNA damage can cause cell cycle arrest and induce apoptosis, which may extensively destroy the potential of progenitor cells and, consequently, impair heart development7). It has been well documented that environmental pollutants, including PM2.5, have the potential to attack DNA through oxidative stress mechanisms8,9. Both human and zebrafish cardiac development are sensitive to oxidative stress10,11,12. 8-OHdG is an oxidative DNA damage marker, and &#947;H2AX signal is a marker of DNA double strand breaks. N-acetyl-L-cysteine (NAC), a synthetic precursor of intracellular cysteine and glutathione, is widely used as an anti-oxidative compound. In this study, we use NAC to investigate the role of oxidative stress in PM2.5- induced DNA damage13.
Zebrafish as a model vertebrate has been widely used to study cardiac development and human cardiovascular diseases because mechanisms of cardiac development are highly conserved among vertebrates14,15. The advantages of using zebrafish as a model include their small size, strong reproductive ability, and low feeding cost. Of particular interest to these studies, zebrafish embryos do not depend on the circulatory system during early development and can survive severe heart malformation14. Moreover, their transparency allows the entire body to be directly observed under a microscope. Thus, zebrafish embryos provide an outstanding opportunity for assessing the molecular mechanisms involved in the induction of cardiac developmental toxicity as a result of exposure to various environmental chemicals5,16,17. We have previously reported that PM2.5-induced oxidative stress leads to DNA damage and apoptosis, resulting in heart malformations in zebrafish18. In this study, we provide a detailed protocol for investigating PM2.5-induced DNA damage in the heart of zebrafish embryos.

<table><tbody><tr><td>8-OHdG Antibody</td><td>Santa Cruz Biotechnology, USA</td><td>sc-66036</td><td>Primary antibody</td></tr><tr><td>Analytical balance</td><td>Sartorius,China</td><td>BSA124S</td><td></td></tr><tr><td>BSA</td><td>Solarbio,Beijing,China</td><td>SW3015</td><td>For blocking</td></tr><tr><td>DAPI</td><td>Abcam, USA</td><td>ab104139</td><td>For nuclear counterstain.</td></tr><tr><td>DMSO</td><td>Solarbio,Beijing,China</td><td>D8371</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Olympus, Japan</td><td>IX73</td><td>For imaging fluorescence signals/</td></tr><tr><td>Goat Anti-Rabbit IgG Cy3</td><td>Carlsbad,USA</td><td>CW0159</td><td>Secondary antibody</td></tr><tr><td>Goat Anti-Rabbit IgG FITC</td><td>Carlsbad,USA</td><td>RS0003</td><td>Secondary antibody</td></tr><tr><td>N-Acetyl-L-cysteine(NAC)</td><td>Adamas-Beta, Shanghai, China</td><td>616-91-1</td><td></td></tr><tr><td>Orbital shaker</td><td>QILINBEIER,China</td><td>TS-1</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma,China</td><td>P6148</td><td>Make 4% paraformaldehyde for fixation.</td></tr><tr><td>Phosphate Buffered Saline</td><td>HyClone,USA</td><td>SH30256.01</td><td>Prepare 0.1% Tween in PBS for washing.</td></tr><tr><td>PM2.5 sampler</td><td>TianHong,Wuhan, China</td><td>TH-150C</td><td>For 24-hr uninterrupted PM2.5 sampling.</td></tr><tr><td>Re-circulating aquaculture system</td><td>HaiSheng,Shanghai,China</td><td></td><td>The zebrafish was maintained in it.</td></tr><tr><td>Soxhlet extractor</td><td>ZhengQiao,Shanghai, China</td><td>BSXT-02</td><td>For organic components extraction.</td></tr><tr><td>Stereomicroscope</td><td>Nikon,Canada</td><td>SMZ645</td><td>For heart dissection from zebrafish embryos.</td></tr><tr><td>Tricaine methanesulfonate (MS222)</td><td>Sigma,China</td><td>E10521</td><td>To anesthetize zebrafish embryos</td></tr><tr><td>Tween 20</td><td>Sigma,China</td><td>P1379</td><td></td></tr><tr><td>&amp;gamma;H2AX Antibody</td><td>Abcam, USA</td><td>ab26350</td><td>Primary antibody</td></tr></tbody></table>

using immunofluorescence to detect pm2.5-induced dna damage in zebrafish embryo hearts

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2&#39;deoxygenase (8-OHdG) is a marker of oxidative DNA damage and &#947;H2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and &#947;H2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 &#956;g/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 &#956;M) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and &#947;H2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and &#947;H2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

This immunofluorescence assay is a sensitive and specific method for measuring protein expression changes in the hearts of zebrafish embryos exposed to environmental chemicals.
In this representative analysis, embryos exposed to PM2.5 in the absence or presence of the antioxidant NAC were evaluated for the presence the presence of heart malformations (Figure 1). As observed, EOM from PM2.5 caused a significant increase in cardiac teratogenesis, such as pericardial edema, altered looping, and decreased size, compared to DMSO control-treated hearts. The heartbeat rate was also significantly decreased in embryos exposed to EOM (Figure 1B). The addition of NAC significantly attenuated EOM-induced heart defects (Figure 1).
Here the immunofluorescence assay was used to measure 8-OHdG and &#947;H2AX expression in zebrafish embryo to evaluate the extent of DNA damage in EOM-treated tissues. As shown in Figure 2, the levels of 8-OHdG and &#947;H2AX expression were significantly increased in the hearts of zebrafish embryos treated with EOM group compared to the control, DMSO-treated hearts, indicating an increase in oxidative DNA damage and DNA double strand breaks, respectively. Furthermore, the EOM-induced DNA damage was partially counteracted by NAC supplementation (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62021/62021fig01.jpg" /> 
Figure 1. Cardiac defects of zebrafish embryos at 72 hpf. (A) Images of zebrafish embryos at 72 hpf. Dotted lines indicate atria (red) or ventricles (blue). Scale bar, 200 &#956;m. (B) Heart malformation and heartbeat rates. Results are presented as mean &#177; SEM. At least 50 embryos in each group were examined. EOM: EOM at 5 mg/L; NAC: NAC at 0.25 &#956;M.. **,P &#60; 0.01; ***, p&#60;0.001 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62021/62021fig01large.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/62021/62021fig02.jpg" /> 
Figure 2.&#160;DNA damage in the heart of zebrafish embryos at 72 hpf. A) Immunofluorescence staining. Scale bar, 100 &#956;m. B) Quantitative results. Results were presented as mean &#177; SEM. At least 15 hearts from each group were examined. EOM: EOM at 5 mg/L; NAC: NAC at 0.25 &#956;M. **; P &#60; 0.01; ***; p&#60;0.001.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62021/62021fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts at JoVE.com

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

3.7K Views.  Soochow University. This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Video: Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Biology

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 ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Using Immunofluorescence to Detect PM_2.5-induced DNA Damage in Zebrafish Embryo Hearts

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Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Single Cell Fate Mapping in Zebrafish

Immunostaining of Dissected Zebrafish Embryonic Heart

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Analysis of Oxidative Stress in Zebrafish Embryos

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos