Name: using immunofluorescence to detect pm2.5-induced dna damage in zebrafish embryo hearts
Uploaded: 2021-02-15T13:00:00
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.
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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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#947;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 PM<s...

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. ...

This method can be used to accurately and easily detect the expression of target proteins in the zebrafish heart. The change in the target protein are directly detected by measuring immunofluorescence. Perform zebrafish embryo collection and treatment, as described in the text manuscript.At 72 hours post-fertilization, transfer the embryos to glass slides, and observe them under a stereo microscope. Record heart malformations, such as pericardial edema, altered looping and decreased size. Calculate malformation rates and analyze differences between groups using one way ANOVA, followed by Turkey's multiple comparison test.After anesthetizing the embryos with 0.6 milligrams per milliliter MS222, record heartbeats for 30 seconds, and quantify heart rates using Image J software. Carefully dissect hearts from the zebra fish embryos with a disposable syringe needle under a stereo microscope. Use a hydrophobic barrier pen to draw a circle on a clean glass slide.Add 50 microliters of 4%paraformaldehyde to 1.25 microliters of PBS to make a fixative solution, then place three dissected hearts into one hydrophobic barrier circle, and incubate for 20 minutes at room temperature. Decant the solution under the microscope and dry the samples at room temperature for at least five minutes. Wash the slides three times in PBST for five minutes per wash.Add 50 microliters of BSA to 1, 000 microliters of PBST to obtain a 5%BSA solution, and incubate the slides in a humid chamber for one hour to block non-specific antibody binding. Decant the solution and wash the samples three times with PBS for five minutes per wash. Dilute two microliters of mouse monoclonal antibody against 8-hydroxydeoxyguanosine and two microliters of rabbit polyclonal antibody against gamma-H2AX in 296 microliters of PBST to obtain a working primary antibody cocktail solution.Incubate the heart samples with 50 microliters of the primary antibody cocktail solution in a humidified chamber for at least one hour at room temperature or overnight at four degrees Celsius. Decant the solution and wash the samples three times with PBST for five minutes per wash. Dilute one microliter of FITC-labeled goat anti-mouse secondary antibody and one microliter of psy-3 goat anti-rabbit secondary antibody in 500 microliters of PBST to obtain a working secondary antibody cocktail solution.Incubate the samples with the secondary antibodies for one hour at room temperature in the dark. Decant the solution and wash the samples three times with PBS for five minutes per wash. Add 20 microliters of DAPI to the samples for nuclear staining, and incubate them for 30 minutes at room temperature.Apply a cover slip to the slide and seal it with nail polish to prevent drying and movement. Image the samples under a fluorescence microscope and quantify the fluorescent signal of the heart area using ImageJ software. Calculate the relative changes using the average of the DMSO control samples and determine the statistical significance of the data.Embryos exposed to PM 2.5 in the absence or presence of the antioxidant, N-acetyl-L-cysteine, or NAC, were evaluated for the presence of heart malformations. EOM from PM 2.5 cost a significant increase in cardiac teratogenesis, such as pericardial edema, altered looping and decreased size, compared to DMSO-control treated hearts. The heart rate was also significantly decreased in embryos exposed to EOM.Addition of NAC attenuated EOM-induced heart defects. An immunofluorescence assay was used to evaluate the extent of DNA damage in EOM-treated tissues. The levels of 8-hydroxydeoxyguanosine and gamma-H2AX expression were significantly increased in the hearts of zebrafish embryos treated with EOM, indicating an increase in oxidative DNA damage and DNA double strand breaks, respectively.The EOM-induced DNA damage was partially counteracted by NAC supplementation. When attempting this protocol, take care when washing the samples with PBS, because the zebrafish heart may peel off the slide.

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