Research support was provided by a UCR Graduate Division Fellowship to SAB, a NRSA T32 Training Program Fellowship (T32ES018827) to SAB, and a National Institutes of Health grant (R01ES027576) and USDA National Institute of Food and Agriculture Hatch Project (1009609) to DCV.

Adult breeders were handled and treated in accordance with an Institutional Animal Care and Use Committee (IACUC)-approved animal use protocol (#20180063) at the University of California, Riverside.
1. Zebrafish embryo collection and chemical exposure
<ol>
	<li>Add in-tank breeding traps to tanks containing sexually mature and reproductively viable adult male and female zebrafish. Add at least three traps per 6 L tank at least 12 h prior to collection at ~9:00 AM, which is t...

<ol>
	<li>Zhang, H. -. Y., Xiong, J., Qi, B. -. L., Feng, Y. -. Q., Yuan, B. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+existence+of+5-hydroxymethylcytosine+and+5-formylcytosine+in+both+DNA+and+RNA+in+mammals.">The existence of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA in mammals.</a> Chemical Communications. 52 (4), 737-740 (2016).</li><li>Huang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+determination+of+the+oxidation+products+of+5-methylcytosine+in+RNA.">Formation and determination of the oxidation products of 5-methylcytosine in RNA.</a> Chemical Science. 7 (8), 5495-5502 (2016).</li><li>Avila-Barnard, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tris(1,3-dichloro-2-propyl)+phosphate+disrupts+the+trajectory+of+cytosine+methylation+within+developing+zebrafish+embryos.">Tris(1,3-dichloro-2-propyl) phosphate disrupts the trajectory of cytosine methylation within developing zebrafish embryos.</a> Environmental Research. 211, 113078 (2022).</li><li>Dasgupta, S., Cheng, V., Volz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+zebrafish+embryos+to+reveal+disruptions+in+dorsoventral+patterning.">Utilizing zebrafish embryos to reveal disruptions in dorsoventral patterning.</a> Current Protocols. 1 (6), 179 (2021).</li><li>Vliet, S. M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal-to-zygotic+transition+as+a+potential+target+for+niclosamide+during+early+embryogenesis.">Maternal-to-zygotic transition as a potential target for niclosamide during early embryogenesis.</a> Toxicology and Applied Pharmacology. 380, 114699 (2019).</li><li>Kupsco, A., Dasgupta, S., Nguyen, C., Volz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+alterations+in+DNA+methylation+precede+Tris(1,3-dichloro-2-propyl)phosphate-induced+delays+in+zebrafish+epiboly.">Dynamic alterations in DNA methylation precede Tris(1,3-dichloro-2-propyl)phosphate-induced delays in zebrafish epiboly.</a> Environmental Science &amp; Technology Letters. 4 (9), 367-373 (2017).</li><li>Dasgupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+interplay+among+nuclear+receptor+ligands,+cytosine+methylation,+and+the+metabolome+in+driving+tris(1,3-dichloro-2-propyl)phosphate-induced+epiboly+defects+in+zebrafish.">Complex interplay among nuclear receptor ligands, cytosine methylation, and the metabolome in driving tris(1,3-dichloro-2-propyl)phosphate-induced epiboly defects in zebrafish.</a> Environmental Science &amp; Technology. 53 (17), 10497-10505 (2019).</li><li>McGee, S. P., Cooper, E. M., Stapleton, H. M., Volz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+zebrafish+embryogenesis+is+susceptible+to+developmental+TDCPP+exposure.">Early zebrafish embryogenesis is susceptible to developmental TDCPP exposure.</a> Environmental Health Perspectives. 120 (11), 1585-1591 (2012).</li><li>Geiman, T. M., Muegge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+early+development.">DNA methylation in early development.</a> Molecular Reproduction and Development. 77 (2), 105-113 (2010).</li><li>Wossidlo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxymethylcytosine+in+the+mammalian+zygote+is+linked+with+epigenetic+reprogramming.">5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.</a> Nature Communications. 2 (1), 1-8 (2011).</li><li>Rottach, A., Leonhardt, H., Spada, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation-mediated+epigenetic+control.">DNA methylation-mediated epigenetic control.</a> Journal of Cellular Biochemistry. 108 (1), 43-51 (2009).</li><li>Egger, G., Liang, G., Aparicio, A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+human+disease+and+prospects+for+epigenetic+therapy.">Epigenetics in human disease and prospects for epigenetic therapy.</a> Nature. 429 (6990), 457-463 (2004).</li><li>Ooi, S. K. T., O'Donnell, A. H., Bestor, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cytosine+methylation+at+a+glance.">Mammalian cytosine methylation at a glance.</a> Journal of Cell Science. 122 (16), 2787-2791 (2009).</li><li>Baylin, S. B., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+exploring+the+cancer+epigenome-biological+and+translational+implications.">A decade of exploring the cancer epigenome-biological and translational implications.</a> Nature Reviews Cancer. 11 (10), 726-734 (2011).</li><li>Robertson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+and+chromatin-unraveling+the+tangled+web.">DNA methylation and chromatin-unraveling the tangled web.</a> Oncogene. 21 (35), 5361-5379 (2002).</li><li>Robertson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+and+human+disease.">DNA methylation and human disease.</a> Nature Reviews Genetics. 6 (8), 597-610 (2005).</li><li>Greenberg, M. V. C., Bourc'his, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+roles+of+DNA+methylation+in+mammalian+development+and+disease.">The diverse roles of DNA methylation in mammalian development and disease.</a> Nature Reviews Molecular Cell Biology. 20 (10), 590-607 (2019).</li><li>Yuan, B. -. F., Feng, Y. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+analysis+of+5-methylcytosine+and+its+oxidation+products.">Recent advances in the analysis of 5-methylcytosine and its oxidation products.</a> Trends in Analytical Chemistry. 54, 24-35 (2014).</li><li>Lantz-McPeak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+assay+using+high+content+screening+of+zebrafish+embryos.">Developmental toxicity assay using high content screening of zebrafish embryos.</a> Journal of Applied Toxicology. 35 (3), 261-272 (2015).</li></ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

During this protocol, there are a few steps that are critical. First, when dechorionating embryos, it is important to point the needle away from the tissue of the embryo/yolk sac/cell mass, as these portions of the developing embryo are very fragile and easy to puncture. Second, when transferring labeled embryos to individual wells, use a glass pipette to transfer embryos as they will adhere to a plastic pipette. Third, when performing whole-mount IHC, ensure that the plate is protected from light. Finally, after complet...

Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine1,2. Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used flame retardant in the United States that has been previously demonstrated to alter the trajectory of cytosine methylation following early embryonic exposure from 0.75 hours post-fertilization (hpf) through early gastrulation (6 hpf)3,4,5,6,7,8. Within vertebrates, 5-mC and its modified derivatives are critical for regulating early embryonic development9. Fertilization of an embryo triggers demethylation of parental DNA, followed by maternal mRNA degradation, zygotic genome activation, and remethylation of the zygotic genome9. Biologically relevant processes that utilize cytosine methylation include histone modification, recruitment of transcriptional machinery, RNA methylation, epigenetic reprogramming, and determination of chromatin structure10,11. Cytosine methylation is also conserved among vertebrate species, underscoring the importance of understanding and investigating how aberrant cytosine methylation may affect the trajectory of an organism&#39;s development11. Furthermore, in utero development is sensitive to maternal exposure and has the potential to cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks12,13,14.
Long stretches of cytosine-guanine pairs, or CpG islands, have been the primary foci of investigators that aim to characterize the dynamics of cytosine methylation across the genome15,16,17. Bisulfite-based strategies such as whole-genome bisulfite sequencing, reduced representation bisulfite sequencing, and bisulfite amplicon sequencing represent the gold standard for interrogating cytosine methylation at base-pair resolution. However, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. In addition, sequencing-based approaches do not provide information about spatial localization, which is critical for understanding potentially affected cell types and areas within a developing embryo. Similarly, global DNA methylation assays such as methylation-dependent restriction analysis, 5-mC enzyme-linked immunoassays (ELISAs), and 5-methyl-2&#39;-deoxycytidine (5-mC) liquid chromatography-mass spectrometry (LC-MS) rely on cell or tissue homogenates and, as such, preclude the ability to monitor the localization and magnitude of cytosine methylation over space and time within intact specimens12,18.
Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be widely used as physiologically intact models to uncover xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available antibodies specific to 5-mC, the protocol below describes a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry (IHC), automated high-content imaging, and efficient data processing using programming language prior to statistical analysis.
To current knowledge, this method is the first to monitor 5-mC within intact zebrafish embryos. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5-mL microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>540225</td>
			<td></td>
		</tr>
		<tr>
			<td>10-&#181;L glass microcapillary pipette</td>
			<td>Fisher Scientific</td>
			<td>211762B</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm plastic Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08757100D</td>
			<td></td>
		</tr>
		<tr>
			<td>10x phosphate-buffered saline&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP399500</td>
			<td></td>
		</tr>
		<tr>
			<td>1-mL pipette&#160;</td>
			<td>Fisher Scientific</td>
			<td>13690032</td>
			<td></td>
		</tr>
		<tr>
			<td>250-mL Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>FB501250</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL pipette</td>
			<td>Fisher Scientific</td>
			<td>13690033</td>
			<td></td>
		</tr>
		<tr>
			<td>60-mm glass petri dishes with lids</td>
			<td>Fisher Scientific</td>
			<td>08747A</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Fisher Scientific</td>
			<td>720089</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488-conjugated goat anti-mouse IgG antibody&#160;</td>
			<td>Fisher Scientific</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>BP67110</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>BP2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate&#160;</td>
			<td>Fisher Scientific</td>
			<td>1110016SH</td>
			<td></td>
		</tr>
		<tr>
			<td>In-tank breeding traps</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td>This product is no longer available following acquisition of Aquatic Habitats by Pentair.&#160; Investigators can use standard off-system breeding tanks available from multiple vendors.</td>
		</tr>
		<tr>
			<td>ImageXpress Micro XLS Widefield High-Content Screening System</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Any high-content screening system equipped with transmitted light and FITC filter will be suitable.</td>
		</tr>
		<tr>
			<td>Immunochemistry (IHC) basket</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Manufactured in-house using microcentrifuge tubes with conical portion removed and bottom fitted with mesh, sized for 24- or 48-well plates.</td>
		</tr>
		<tr>
			<td>MetaXpress 6.0.3.1658&#160;</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Any software capable of quantifying total area and integrated intensity of fluorescence will be suitable.</td>
		</tr>
		<tr>
			<td>Microspatula</td>
			<td>Fisher Scientific</td>
			<td>2140115</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-5-mC antibody</td>
			<td>Millipore Sigma</td>
			<td>MABE146</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>BP359-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker&#160;</td>
			<td>Fisher Scientific</td>
			<td>50998290</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm&#160;</td>
			<td>Fisher Scientific</td>
			<td>1337412</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Fisher Scientific</td>
			<td>18612139</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>1368050</td>
			<td></td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>RStudio</td>
			<td>N/A</td>
			<td>RStudio is open-source software and can be downloaded at https://www.rstudio.com.</td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Millipore Sigma</td>
			<td>S3772-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>10450103</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature-controlled incubator&#160;</td>
			<td>Fisher Scientific</td>
			<td>PR505755L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20&#160;</td>
			<td>Fisher Scientific</td>
			<td>P7949-500ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

The overall aim of this protocol is to determine whether a treatment affects the relative abundance of 5-mC by assessing the total area and relative intensity of fluorescence within fixed and labeled zebrafish embryos. After completing the protocol, a fluorescence stereomicroscope can be used to first determine whether the whole-mount IHC was successful. When labeled embryos are observed under a FITC or GFP filter, a positive result is indicated by a positive FITC signal within the embryo, whereas a negative result is in...

Watch this Scientific Journal Video about Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos at JoVE.com

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

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

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

This protocol offers a relatively rapid and cost efficient method, as well as an automated acquisition and analysis thus producing low sensitivity screening of 5-Methylcytosine which is a common epigenetic biomarker. The main advantage of this technique is in the flexibility it offers an experimental design that users can optimize to their model and stage of development. This technique can be used to detect how chemical exposure alters the relative abundance of 5-Methylcytosine within a developing organism which normally could only be done through expensive high trpA methods such as sequencing.This method is useful to any research which aims to utilize NCG methods to understand epigenetic alterations of 5-Methylcytosine in response to environmental stimuli. Begin by preparing a fresh exposure solution on the morning of collection. Add five milliliters of particulate free system water to a 60 millimeter glass Petri dish.Then use a 10 microliter micro capillary glass pipette to transfer 10 microliters of dimethyl sulfoxide or chemical stock solution to system water. Swirl the glass Petri dish to ensure the stock solution dissipates. Add another five milliliters of system water to the glass Petri dish and swirl the dish to homogenize the exposure solution.Cap the glass dishes with glass lids to minimize evaporation. Next, use an RO water-filled squirt bottle to transfer the embryos from the fish net into a 100 millimeter dish. Randomly sort 50 live embryos at 0.75 hours post fertilization and remove excess RO water.After the exposure duration has ended, remove the embryos from the incubator. Transfer all living embryos to a 1.5 milliliter micro centrifuge tube. Aspirate residual exposure solution without aspirating any embryos.Add 500 microliters of chilled 4%paraformaldehyde and mix the embryos by inversion several times. Allow the embryos to fix and 4%paraformaldehyde overnight at four degrees celsius. After fixing the embryos aspirate the 4%paraformaldehyde, respend the embryos in 1X PBS, and mix by gentle pipetting for 30 seconds.Using a glass pipette, carefully transfer fixed embryos to an RO water-filled glass dish. Gently swirl for 30 seconds and repeat this wash once. Under a stereo microscope set at five times magnification decorenate the embryos using syringe needles.Using the needle tip, puncture the chorion and gently peel it away from the yolk and cell mass. After the embryos have been decorenated, use a glass micro capillary pipette to transfer up to 25 intact embryos into one immunohistochemistry basket. Place the IHC basket into a 24 well plate.Then aspirate the water from each well. Resuspend the embryos in 500 microliters of blocking buffer per well, wrap the plate with paraform and aluminum foil to protect it from light. Incubate the plate at four degrees Celsius for four hours on an orbital shaker at 100 rpm.After incubation, aspirate the blocking buffer from each well. Replace it with 500 microliters of primary antibody solution. Remember to incubate a subset of vehicle control embryos with the blocking buffer to account for background noise.Rewrap the plate and paraform and aluminum foil and allow the plate to incubate at four degree Celsius overnight on an orbital shaker. The following day remove the primary antibody solution from each well and replace it with 1x PBST. Wash each well thrice with 1x PBST for five minutes per wash on an orbital shaker.Aspirate 1x PBST from each well and place the IHC basket into a glass Petri dish filled with RO water. Under a standard stereo microscope randomly sort intact embryos into a 96 well plate ensuring one embryo per well. Then remove all RO water from individual wells and add 200 microliters of 1x PBS.Centrifuge the plate for three minutes at one G.Image the embryos with a two times objective under transmitted light and FITC. Select autofocus to ensure the focus of the camera is on the bottom of the plate and offset by the bottom thickness. Select a FITC wavelength with an exposure duration of 100 milliseconds and set the auto focus to laser with a Z offset.Observe and confirm proper image acquisition and several wells prior to commencing plate acquisition. After image acquisition, carry out data analysis using the high content screening system and a custom automated image analysis procedure. Select each embryo on the 96 well plate to be analyzed for total area and integrated intensity of fluorescence.Then tabulate the data points and export them from the high content screening system by going to measure and selecting open data log to export the data to a spreadsheet. Upload the exported spreadsheet to the deployer package to sort and summate data for each well and filter by well number. Then use the right Excel package to export the summated data into a spreadsheet.The distribution and integrated intensity of 5-Methylcytosine specific total area were assessed for Zebrafish embryos at six hours post fertilization. The x-axis shows exposure to either DMSO as a vehicle or TDCIPP as the positive control. The Y-axis denotes relative fluorescence.The thresholds were significantly different from the vehicle treated embryos as shown by the total area of 5-Methylcytosine detected within embryos and the integrated intensity within that same area indicating maximum signal to noise ratio. It is extremely important to maintain the integrity of the yoke and cell mass for proper IHC detection. This method is relatively insensitive and only offers relative abundance RFU data.If quantification is required one can follow up with a more targeted approach.

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1.3K Views.  University of California, Riverside. This protocol offers a relatively rapid and cost efficient method, as well as an automated acquisition and analysis thus producing low sensitivity screening of 5-Methylcytosine which is a common epigenetic biomarker. The main advantage of this technique is in the flexibility it offers an experimental design that users can optimize to their model and stage of development. This technique can be used to detect how chemical exposure alters the relative abundance of 5-Methylcytosine within a devel...