Name: Detection of Global DNA Methylation in the Hearts of Zebrafish Larvae (Video) | JoVE Journal
Uploaded: 2026-04-10T12:53:00.000+00:00
Duration: 5 min 7 s
Description: Learn an immunofluorescence method to quantify global DNA methylation in zebrafish embryonic hearts using 5-Azacytidine exposure. This sensitive protocol enables epigenetic analysis of cardiac development.

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5 minutes

This research was funded by the National Natural Science Foundation of China (Grant number: 81870239) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

The AB strain wild-type zebrafish were acquired from the National Zebrafish Resource Center (Wuhan, China). All animal procedures were reviewed and approved by the Animal Protection Institute of the Ethics Committee of Soochow University. The reagents and the equipment used are listed in the Table of Materials.
1. Zebrafish embryo treatment and heart isolation
<ol>
	<li>Maintain adult zebrafish in a flow-through aquaculture system under controlled conditions (28.5 &#176;C &#177; 0.5 &#176;C, 14-h light/10-h dark photoperiod).</li>
	<li>Transfer sexually mature fish (maintaining a 2:1 female-to-male ratio) to sterilized breeding tanks.</li>
	<li>Collect embryos the following day and rinse with system water.</li>
	<li>Select developmentally synchronized embryos and randomly assign them to three experimental groups (50 embryos per group) in 7 cm glass culture dishes.</li>
	<li>Dissolve 5-AzaC in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. Aliquot and store the stock solution at -20 &#176;C.Immediately before use, dilute the 5-AzaC stock solution in embryo medium to obtain the desired final working concentrations of 25 &#181;M and 50 &#181;M.
	<ol>
		<li>Prepare a vehicle control solution by diluting DMSO in embryo medium to a final concentration of 0.1 % (v/v). Add 5-AzaC or DMSO to the glass dishes containing embryos at 2 hpf. Renew the exposure solutions every 24 h and discard dead embryos.</li>
	</ol>
	</li>
	<li>At 72 hpf, assess heart malformations (such as string-like hearts, elongated hearts, and pericardial edema) by observing embryos under a stereo microscope. For cardiac function analysis, acquire videos using a stereo microscope, and process heart rate (HR) using DanioScope Software.</li>
	<li>Wash the larvae three times for 5 min each. Anesthetize the larvae with 2.5 mg/L MS-222 and place them on a glass slide in a small drop of embryo medium to maintain tissue hydration.</li>
	<li>Use a 25 G disposable syringe needle (0.3 mm outer diameter) to gently make a small incision in the ventral thoracic region of the larvae (anterior to the yolk sac) under a stereo microscope (10&#215;&#8211;40&#215; magnification).</li>
	<li>Dissect the pericardial sac and separate the heart from the surrounding somatic tissue using the needle tip; Immediately transfer the isolated heart to PBST using a pipette for subsequent fixation.</li>
</ol>
2. Immunofluorescence staining
<ol>
	<li>Transfer 3 isolated hearts into a hydrophobic barrier pen circle on an adhesive slide using a pipette.</li>
	<li>Add 45 &#181;L of 4% Paraformaldehyde (PFA) and fix for 30 min at Room temperature (RT). Wash the samples twice with Phosphate Buffered Saline with Tween 20 (PBST, 1x PBS with 0.1% Tween-20), using 50 &#181;L per wash for 5 min each.</li>
	<li>Permeabilize by adding 45 &#181;L of 0.3% Triton X-100 and incubating for 35 min at RT; wash 3&#215; 5 min with PBST.</li>
	<li>Add 45 &#181;L of Proteinase K (10 mg/mL) and incubate for 30 min, followed by 3&#215; 5 min PBST washes.</li>
	<li>Treat with 45 &#181;L of 4M Hydrochloric Acid (HCl) for 30 min. 
	NOTE: This step is crucial for effective DNA denaturation.</li>
	<li>Neutralize with Tris (Hydroxymethyl) Aminomethane Hydrochloride (Tris-HCl) buffer (pH 8.5) for 20 min; follow with 3&#215; 5 min PBST washes.</li>
	<li>Block nonspecific binding by incubating with 45 &#181;L of 10% goat serum for 1 h at RT; wash 3&#215; 5 min PBST.</li>
	<li>Incubate with anti-5-Methylcytosine (anti-5mC) antibody (diluted 1:300 in 5 % goat serum) overnight at 4 &#176;C.</li>
	<li>Remove primary antibody and wash 3&#215; 10 min with PBST.</li>
	<li>Incubate with 45 &#181;L of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500) for 1 h at RT in the dark; wash 3&#215; 10 min with PBST.</li>
	<li>Co-stain nuclei with 10 &#181;L of 4&#39;,6-Diamidino-2-phenylindole dihydrochloride (DAPI).</li>
	<li>Cover the slides with a coverslip and keep them in the dark for 1 h.</li>
	<li>Acquire images using a standard inverted fluorescence microscope.</li>
	<li>Quantify the fluorescence intensity using ImageJ software.</li>
</ol>
3. Image acquisition
<ol>
	<li>Acquire images using an inverted epifluorescence microscope.</li>
	<li>Use a 20&#215; objective lens to capture the entire embryonic heart structure.</li>
	<li>Image the DAPI channel (excitation/emission: ~358/461 nm) and the Alexa Fluor 488 channel (excitation/emission: ~495/519 nm) sequentially using standard filter sets to avoid crosstalk.</li>
	<li>Maintain identical exposure times, gain, and light intensity settings for all samples within the same experiment. For example, the exposure time for the Alexa Fluor 488 (5mC) channel was set to 200 m.</li>
	<li>Save the images in a lossless format.</li>
</ol>
4. ImageJ quantification
<ol>
	<li>Open the multi-channel image file. Select the green channel (corresponding to Alexa Fluor 488) for 5mC signal analysis.</li>
	<li>Utilize the freehand selection tool to delineate a Region of Interest (ROI) encompassing the entire heart tissue.</li>
	<li>With the heart ROI active, measure the Mean Gray Value (Analyze &#62; Measure) and record this value.</li>
	<li>Relocate the same ROI to an adjacent, tissue-free area of the image and measure the Mean Gray Value again to obtain the background value.</li>
	<li>Calculate the corrected fluorescence intensity by subtracting the background value from the heart ROI measurement.</li>
	<li>Repeat the procedure for all samples and perform subsequent analysis.</li>
</ol>
5. Statistical analyses
<ol>
	<li>Conduct statistical analyses using GraphPad Prism 8 software and report data as mean &#177; SEM.</li>
	<li>Use one-way ANOVA following Dunnett&#39;s multiple comparisons test to assess statistical significance.</li>
</ol>

<ol><li>Schmitz, R. J., Lewis, Z. A., Goll, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+methylation%3A+Shared+and+divergent+features+across+eukaryotes%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">DNA methylation: Shared and divergent features across eukaryotes.</a> Trends Genet. 35 (11), 818-827 (2019).</li>
<li>Zhang, X., Zhang, Y., Wang, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TET+%28ten-eleven+translocation%29+family+proteins%3A+Structure%2C+biological+functions+and+applications%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">TET (ten-eleven translocation) family proteins: Structure, biological functions and applications.</a> Signal Transduct Target Ther. 8 (1), 1-15 (2023).</li>
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<li>Gilsbach, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+DNA+methylation+orchestrates+cardiomyocyte+development%2C+maturation+and+disease%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.</a> Nat Commun. 5, 5288(2014).</li>
<li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alteration+in+methylation+pattern+of+GATA-4+promoter+region+in+vitamin+A-deficient+offspring%27s+heart%5BTitle%5D)+AND+%22J+Nutr+Biochem%22%5BJournal%5D)">Alteration in methylation pattern of GATA-4 promoter region in vitamin A-deficient offspring's heart.</a> J Nutr Biochem. 24 (7), 1373-1380 (2013).</li>
<li>Chamberlain, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+methylation+is+developmentally+regulated+for+genes+essential+for+cardiogenesis%5BTitle%5D)+AND+%22J+Am+Heart+Assoc%22%5BJournal%5D)">DNA methylation is developmentally regulated for genes essential for cardiogenesis.</a> J Am Heart Assoc. 3 (3), e000976(2014).</li>
<li>Tu, S., Chi, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+models+in+cardiac+development+and+congenital+heart+birth+defects%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Zebrafish models in cardiac development and congenital heart birth defects.</a> Differentiation. 84 (1), 4-16 (2012).</li>
<li>Goll, M. G., Halpern, M. E. DNA methylation in zebrafish. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGoll%2C+MG+%22Prog+Mol+Biol+Transl+Sci%22">Prog Mol Biol Transl Sci</a>. Cheng, X., Blumenthal, R. M. 101, 193-218 (2011).</li>
<li>Bouwmeester, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+embryos+as+a+screen+for+DNA+methylation+modifications+after+compound+exposure%5BTitle%5D)+AND+%22Toxicol+Appl+Pharmacol%22%5BJournal%5D)">Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.</a> Toxicol Appl Pharmacol. 291, 84-96 (2016).</li>
<li>Brown, D. R., Samsa, L. A., Qian, L., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+the+study+of+heart+development+and+disease+using+zebrafish%5BTitle%5D)+AND+%22J+Cardiovasc+Dev+Dis%22%5BJournal%5D)">Advances in the study of heart development and disease using zebrafish.</a> J Cardiovasc Dev Dis. 3 (2), 13(2016).</li>
<li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+immunofluorescence+to+detect+PM2.5-induced+DNA+damage+in+zebrafish+embryo+hearts%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Using immunofluorescence to detect PM2.5-induced DNA damage in zebrafish embryo hearts.</a> J Vis Exp. (168), e62107(2021).</li>
<li>Cavalieri, V., Spinelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+epigenetics+in+zebrafish%5BTitle%5D)+AND+%22Epigenetics+Chromatin%22%5BJournal%5D)">Environmental epigenetics in zebrafish.</a> Epigenetics Chromatin. 10 (1), 46(2017).</li>
<li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PM2.5-induced+extensive+DNA+methylation+changes+in+the+heart+of+zebrafish+embryos+and+the+protective+effect+of+folic+acid%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">PM2.5-induced extensive DNA methylation changes in the heart of zebrafish embryos and the protective effect of folic acid.</a> Environ Pollut. 255 (Pt 3), 113331(2019).</li>
<li>Bonora, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+methylation+estimation+using+methylation-sensitive+restriction+enzyme+bisulfite+sequencing+%28MREBS%29%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">DNA methylation estimation using methylation-sensitive restriction enzyme bisulfite sequencing (MREBS).</a> PLoS One. 14 (4), e0214368(2019).</li>
<li>Kurdyukov, S., Bullock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+methylation+analysis%3A+Choosing+the+right+method%5BTitle%5D)+AND+%22Biology+Basel%22%5BJournal%5D)">DNA methylation analysis: Choosing the right method.</a> Biology Basel. 5 (1), 3(2016).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+techniques+for+DNA-methylation+analysis%5BTitle%5D)+AND+%22TrAC+Trends+Anal+Chem%22%5BJournal%5D)">Development of techniques for DNA-methylation analysis.</a> TrAC Trends Anal Chem. 72, 114-122 (2015).</li>
<li>Treangen, T. J., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repetitive+DNA+and+next-generation+sequencing%3A+Computational+challenges+and+solutions%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Repetitive DNA and next-generation sequencing: Computational challenges and solutions.</a> Nat Rev Genet. 13 (1), 36-46 (2011).</li>
<li>Zhong, S., Li, Z., Jiang, T., Li, X., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunofluorescence+imaging+strategy+for+evaluation+of+the+accessibility+of+DNA+5-hydroxymethylcytosine+in+chromatins%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Immunofluorescence imaging strategy for evaluation of the accessibility of DNA 5-hydroxymethylcytosine in chromatins.</a> Anal Chem. 89 (11), 5702-5706 (2017).</li>
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The authors declare no conflicts of interest.

Research from our group and others has demonstrated that exposure to environmental chemicals can perturb DNA methylation patterns by dysregulating DNMT expression during zebrafish embryonic development13,14. Since 5-AzaC is an effective DNMT inhibitor, it was employed to mimic the effects of environmental chemicals on DNMT activity. The results showed that 5-AzaC treatment significantly reduced global DNA methylation levels in zebrafish larvae hearts. We have con...

DNA methylation is one of the most extensively investigated epigenetic modifications. In vertebrates, DNA methylation predominantly occurs at the 5 position of cytosine (5-methylcytosine, 5mC) within CG dinucleotides. The establishment of DNA methylation Patterns is mediated by de novo DNA methyltransferases (DNMT3A and DNMT3B) and maintained by DNMT1 in conjunction with ubiquitin-like with PHD and RING finger domains 1 (UHRF1)1. The Ten-Eleven Translocation (TET) family of enzymes is essential for the active DNA demethylation process2. DNA methylation within promoter regions or gene bodies typically inversely correlates with gene expression levels3. Global DNA methylation reflects the overall 5mC content and is often assessed by examining methylation levels within repetitive elements4.
DNA methylation plays an essential role during heart development, influencing processes such as atrial and ventricular cell differentiation and lineage specification5. Aberrant DNA methylation patterns have been linked to congenital heart defects (CHD)6,7. Since DNA methylation is sensitive to environmental factors, it may act as an intermediary between xenobiotic exposure and cardiac anomalies.
The zebrafish has been suggested to be a highly suitable vertebrate model for cardiac development research8. Unlike mammals, zebrafish embryos do not rely on a functional cardiovascular system during early development, which facilitates the study of heart formation8. Importantly, the zebrafish DNA methylation system shares considerable similarity with that of mammals9. Zebrafish embryos have also been proposed as a potential screening tool for assessing DNA methylation modifications following compound exposure10. However, the small size of the embryonic zebrafish heart poses a significant challenge in obtaining sufficient DNA for methylation analysis. At 72 hours post-fertilization (hpf), zebrafish hearts have completed cardiogenesis, annularization, and atrioventricular septation, resulting in a structurally distinct and functionally stable biventricular heart that remains in a dynamic state of maturation and remodeling11. Measuring methylation levels at this stage enables identification of critical epigenetic states essential for cardiac structural and functional maturation, as well as for fate determination and maintenance12,13,14.
Several established techniques exist for quantifying&#160;global DNA methylation, including liquid chromatography-tandem mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assays (ELISA), and whole-genome bisulfite sequencing (WGBS)15,16,17. However, all three methods typically require microgram amounts of DNA, which is prohibitive for small tissue samples such as a single zebrafish embryonic heart. Furthermore, WGBS involves bisulfite treatment that induces DNA fragmentation, complicating library preparation and diminishing sequencing complexity18. A sensitive, low-input method needs to be developed for quantifying global 5mC in individual zebrafish embryonic hearts.
5-Azacytidine (5-AzaC) is a DNA methylation inhibitor that reduces genomic DNA methylation levels by promoting demethylation of methylated cytosines5. In this study, a sensitive immunofluorescence approach was established to quantify 5 mC levels in individual zebrafish embryonic hearts after exposure to different concentrations of 5-AzaC.

<table><tbody><tr><td>4% Paraformaldehyde Fix Solution</td><td>Biosharp (Hefei, China)</td><td>BL539A</td><td>This reagent can be employed for the fixation of micro-dissected zebrafish hearts, preserving structural integrity and antigenicity for subsequent histological or immunofluorescence analyses.</td></tr><tr><td>5-AzaC</td><td>Merck (Darmstadt, Germany)</td><td>A2385</td><td>treatment</td></tr><tr><td>Adhesive-Coated Slides for Enhanced Tissue Adherence</td><td>Citotest (Jiangsu, China)</td><td>188105</td><td>Placement of microdissected zebrafish hearts</td></tr><tr><td>AF488-labeled Goat Anti-rabbit IgG</td><td>Beyotime(Shanghai, China)</td><td>A0423</td><td>For Immunofluorescence staining</td></tr><tr><td>Anti-5mC antibody</td><td>Cell Signaling Technology</td><td>28692</td><td>For 5-Methylcytosine (5mC) Immunofluorescence staining</td></tr><tr><td>Cover slip</td><td>Szketone (Suzhou, China)</td><td>GBP-001</td><td>Cover-slip physiological pressure maintenance for zebrafish hearts</td></tr><tr><td>DAPI</td><td>Beyotime(Shanghai, China)</td><td>C1005</td><td>This reagent enables specific nuclear staining through selective targeting of chromatin structures.</td></tr><tr><td>Disposable plastic dropper</td><td>Yinyuebio(Yangzhou, China)</td><td>ZX36C1401</td><td>This device facilitates precise solution replacement within zebrafish embryo culture dishes, enabling controlled manipulation of the embryonic microenvironment while maintaining sterility during experimental interventions.</td></tr><tr><td>DMSO</td><td>Solarbio (Beijing, China)</td><td>D8371</td><td>IS used as a vehicle control.&amp;nbsp;</td></tr><tr><td>Goat Serum</td><td>Beyotime(Shanghai, China)</td><td>&amp;nbsp;C0265</td><td>This reagent is specifically formulated for blocking procedures during immunofluorescence assays, effectively minimizing non-specific antibody binding and reducing background interference through saturation of hydrophobic sites on tissue sections.</td></tr><tr><td>Image J</td><td>NIH(USA)</td><td>1.8.0</td><td>The software is used for fluorescence quantification</td></tr><tr><td>Immunol Fluorescence Staining Secondary Antibody Dilution Buffer</td><td>Beyotime(Shanghai, China)</td><td>P0108</td><td>This reagent is optimized for the preparation and dilution of secondary antibody in immunostaining protocols, ensuring optimal antibody specificity and binding affinity while minimizing non-specific interactions through formulation with carrier proteins and stabilizing agents.</td></tr><tr><td>Immunol Staining Primary Antibody Dilution Buffer</td><td>Beyotime(Shanghai, China)</td><td>P0103</td><td>This reagent is optimized for the preparation and dilution of primary antibodies in immunostaining protocols, ensuring optimal antibody specificity and binding affinity while minimizing non-specific interactions through formulation with carrier proteins and stabilizing agents.</td></tr><tr><td>Inverted Fluorescence Microscope&lt;br/&gt; &amp;nbsp;</td><td>Olympus Corporation(Japan)</td><td>TS2-FL</td><td>Fluorescence image acquisition</td></tr><tr><td>Noldus</td><td>Noldus Information Technology(Netherlands)</td><td>DanioScope Software</td><td>The software is used for heart rate collection and other functions.</td></tr><tr><td>Stereo Dissection Microscope</td><td>Optech(Toronto, Canada)</td><td>YZ39 M63</td><td>This instrument is employed for the micro-dissection of zebrafish hearts.</td></tr><tr><td>Stereo Fluorescence Microscope</td><td>Nikon(Japan)</td><td>SMZ18</td><td>This instrument enables concurrent visualization of cardiac teratology and quantitative recording of heart rate in zebrafish.</td></tr><tr><td>TritonX-100</td><td>Solarbio (Beijing, China)</td><td>P0096</td><td>This reagent enables effective permeabilization for immunostaining protocols, which facilitates epitope unmasking of antigens and accessibility to nucleic acid targets. By compromising membrane integrity through selective disruption of lipid bilayers, it significantly enhances intracellular penetration of antibodies, probes, and labeling molecules, thereby ensuring robust detection sensitivity and analytical reliability.</td></tr><tr><td>Tween-20</td><td>Beyotime(Shanghai, China)</td><td>ST825</td><td>Commonly Utilized Non-Ionic Detergents</td></tr></tbody></table>

detection of global dna methylation in the hearts of zebrafish larvae

Detection of Global DNA Methylation in the Hearts of Zebrafish Larvae

DNA methylation is one of the most extensively studied epigenetic modifications and&#160;plays an essential role in cardiac development. The zebrafish is a popular model for studying heart development due to its genetic similarity to mammals and transparent embryos. However, its small size presents challenges in obtaining sufficient DNA for methylation analysis. In this study, zebrafish embryos were exposed to 5-Azacytidine (5-AzaC) at concentrations of 0 &#181;M, 25 &#181;M, and 50 &#181;M from 2&#8211;72 hours post-fertilization (2&#8211;72 hpf). Hearts were isolated from whole embryos, fixed, and permeabilized. The samples were then treated with proteinase K and HCl to facilitate access to DNA, followed by probing with an anti-5-methylcytosine (anti-5 mC) antibody. After incubation with a secondary antibody, the hearts were co-stained with DAPI. Fluorescence images were captured using a microscope and analyzed with ImageJ. Results demonstrated that 5-AzaC treatment significantly reduced global DNA methylation levels in zebrafish embryonic hearts. In conclusion, we present a sensitive immunofluorescence method to detect global DNA methylation levels within a single zebrafish larvae heart. This technique can be used to evaluate DNA methylation changes induced by environmental pollutants and to explore their impacts on heart development.

As shown in Figure 1A, 5-AzaC at both tested concentrations did not significantly affect the embryo survival rate and hatching rate compared to the DMSO control group. However, 5-AzaC exposure increased cardiac malformation rates in a dose-dependent manner compared with DMSO controls (Figure 1B). Heartbeat rates were decreased to a similar level in the two 5-AzaC treatment groups (Figure 1A).
Hearts were ...

This study establishes a sensitive immunofluorescence-based imaging method to quantify global DNA methylation in individual zebrafish embryonic hearts. This approach overcomes the challenge of limited tissue material and bypasses the need for bisulfite conversion.

DNA methylation is one of the most extensively studied epigenetic modifications and&#160;plays an essential role in cardiac development. The zebrafish is ...

In this study, we developed the sensitive immune fluorescence approach to quantify 5-MZ in zebrafish embryonic hearts. To begin, collect the hearts isolated from zebrafish embryos exposed to 0, 25, or 50 micromolar 5-Azacytidine. Using a pipette, transfer three hearts into the circular area defined by a hydrophobic barrier pen on an adhesive microscope slide.Add 45 microliters of 4%paraformaldehyde and fix the samples for 30 minutes at room temperature. Wash the samples two times with PBS containing Tween 20, using 50 microliters per wash for five minutes each. Next, add 45 microliters of 0.3%Triton X-100 to permeablize the samples and incubate for 35 minutes at room temperature.Wash the samples three times for five minutes each with PBS containing Tween 20. Add 45 microliters of proteinase K and incubate for 30 minutes. Then wash three times with PBS as demonstrated earlier.Treat the samples with 45 microliters of four molar hydrochloric acid for 30 minutes to ensure effective DNA denaturation. Neutralize the samples with a Tris-HCI buffer for 20 minutes. Then wash with PBS containing Tween 20 three times.Incubate the samples with 45 microliters of 10%goat serum for one hour at room temperature to block non-specific binding. Wash the samples three times for five minutes each with PBS containing Tween 20. Incubate the samples with anti-5-methylcytosine antibody overnight at four degrees Celsius.On the following day remove the primary antibody. Wash the samples three times for 10 minutes each with PBS containing Tween 20. Incubate the samples with 45 microliters of Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G for one hour at room temperature in the dark.After three washes with PBS Tween 20, add 10 microliters of DAPI to costain the nuclei. Cover the samples with a cover slip and keep the slides in the dark for one hour. Acquire images using a standard inverted fluorescence microscope.Use a 20x objective lens to capture the entire embryonic heart structure. Image the DAPI channel and Alexa Fluor 488 channel sequentially using standard filter sets to avoid crosstalk. Maintain identical exposure times, gain, and light intensity settings for all samples within the same experiment.Save the images in a lossless format. 5-Azacytidine at both tested concentrations did not significantly affect the embryo survival rate and hatching rate compared with DMSO controls. Heartbeat rates were decreased to a similar level in the two 5-Azacytidine treatment groups compared with DMSO controls.Exposure to 5-Azacytidine at 25 millimolar and 50 millimolar increased cardiac malformation rates compared with DMSO controls. 5-Azacytidine treatment significantly reduced global deoxyribonucleic acid methylation levels in the hearts of zebrafish larvae compared with DMSO controls. Our protocol enables quantitative analysis of DNA methylation and the organ level during early developmental stages.It is essential to ensure complete DNA denaturation and rigorous washing procedures to avoid weak signal or high background. This method can be employed in future research to examine the impact of environmental chemicals on alterations in DNA methylation throughout cardiac development.

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Research

JoVE Journal

Developmental Biology

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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

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.

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

The study aims to develop technology for anesthesia-free heartbeat measurements in moving zebrafish. Our approach combines shortwave-infrared imaging and machine-learning-based tracking of the heart. It is a non-invasive, label-free, and user-friendly technique that suits a wide range of studies on the zebrafish model.

Anesthesia-free Heartbeat Measurements in Freely Moving Zebrafish

This protocol describes a low-cost light microscopy method to assess cardiac morphology and function in zebrafish embryos, enabling reproducible evaluation of developmental cardiotoxicity without the need for advanced imaging systems.

Functional Cardiac Imaging in Zebrafish Embryos Using Standard Microscopy and Video Analysis: Applications in Environmental and Biomedical Research

Learn an immunofluorescence method to quantify global DNA methylation in zebrafish embryonic hearts using 5-Azacytidine exposure. This sensitive protocol enables epigenetic analysis of cardiac development.

Detection of Global DNA Methylation in the Hearts of Zebrafish Larvae

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