Name: rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos
Uploaded: 2022-08-18T14:00:00
Description: Watch this Scientific Journal Video about Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos at JoVE.com

研究支持由 UCR 研究生部奖学金提供给 SAB、NRSA T32 培训计划奖学金 （T32ES018827） 提供给 SAB 以及美国国立卫生研究院赠款 （R01ES027576） 和美国农业部国家粮食和农业研究所孵化项目 （1009609） 提供给 DCV。

成年饲养员按照加州大学河滨分校机构动物护理和使用委员会 （IACUC） 批准的动物使用协议 （#20180063） 进行处理和治疗。
1. 斑马鱼胚胎采集和化学暴露
<ol><li>在含有性成熟和生殖能力强壮的成年雄性和雌性斑马鱼的水箱中添加水箱内繁殖陷阱。在~9：00 AM收集前至少12小时，每个6 L水箱至少添加三个陷阱，这是水箱内受精和产卵的大致时间。</li>
	<li>在收集的?...

<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>

在此协议中，有几个步骤至关重要。首先，在对胚胎进行去皮时，重要的是将针头指向远离胚胎/卵黄囊/细胞团的组织，因为发育中的胚胎的这些部分非常脆弱且易于刺穿。其次，将标记的胚胎转移到单个孔中时，请使用玻璃移液管转移胚胎，因为它们会粘附在塑料移液器上。第三，在进行整体安装IHC时，请确保板免受光照。最后，在完成全安装IHC方案后，在成像前让板在4°C的1x PBS中孵育过夜，?...

酶修饰驱动胞嘧啶的活性甲基化和去甲基化为5-甲基胞嘧啶（5-mC），随后将5-mC氧化为5-羟甲基胞嘧啶、5-甲酰胞嘧啶和5-羧基胞嘧啶1，2。磷酸三（1，3-二氯-2-丙基）酯（TDCIPP）是美国广泛使用的阻燃剂，先前已被证明可以改变受精后0.75小时（hpf）到早期原肠胚形成（6 hpf）的早期胚胎暴露后胞嘧啶甲基化的轨迹3，4，5，6，7，8.在脊椎动物中，5-mC及其修饰的衍生物对于调节早期胚胎发育至关重要9。胚胎受精触发亲本DNA的去甲基化，随后是母体mRNA降解，合子基因组激活和合子基因组的再甲基化9。利用胞嘧啶甲基化的生物学相关过程包括组蛋白修饰、转录机制募集、RNA 甲基化、表观遗传重编程和染色质结构的测定10，11。胞嘧啶甲基化在脊椎动物物种中也是保守的，强调了理解和研究异常胞嘧啶甲基化如何影响生物体发育轨迹的重要性11。此外，子宫内发育对母体暴露敏感，并有可能引起不良后果，例如直接表型后果、对成人疾病易感性的长期影响以及遗传表观遗传标记的跨代效应12，13，14。
长段的胞嘧啶-鸟嘌呤对或CpG岛一直是研究人员的主要焦点，旨在表征整个基因组中胞嘧啶甲基化的动力学15，16，17。基于亚硫酸氢盐的策略，如全基因组亚硫酸氢盐测序、减少代表性亚硫酸氢盐测序和亚硫酸氢盐扩增子测序，代表了在碱基对分辨率下询问胞嘧啶甲基化的金标准。然而，基于测序的方法成本高昂，因此无法监测各个发育阶段的胞嘧啶甲基化、每种化学物质的多种浓度以及每次治疗复制胚胎的能力。此外，基于测序的方法不能提供有关空间定位的信息，这对于了解发育中的胚胎中可能受影响的细胞类型和区域至关重要。同样，全球 DNA 甲基化测定，例如甲基化依赖性限制性分析、5-mC 酶联免疫测定 （ELISA） 和 5-甲基-2'-脱氧胞苷 （5-mC） 液相色谱-质谱 （LC-MS） 依赖于细胞或组织匀浆，因此，无法监测完整标本12，18 内胞嘧啶甲基化随空间和时间的定位和大小。
由于自动化 体内 成像、遗传操作、快速宫 外 发育时间和胚胎发生过程中的饲养易于，斑马鱼胚胎继续被广泛用作生理上完整的模型，以揭示异生素介导的途径，这些途径在早期胚胎发育期间导致不良结果。因此，使用市售的针对5-mC的抗体，以下方案描述了一种经济高效的策略，通过利用全卡口免疫组织化学（IHC）、自动高内涵成像和统计分析前使用编程语言的高效数据处理，对单个完整斑马鱼胚胎中的胞嘧啶甲基化进行快速有效的时空监测。
就目前所知，这种方法是第一个监测完整斑马鱼胚胎内5-mC的方法。该方法能够检测细胞团内的DNA甲基化，并且还能够在母体到合子过渡期间检测卵黄定位母体mRNA的胞嘧啶甲基化。总体而言，该方法将有助于快速鉴定在表观遗传重编程过程中有可能原 位 破坏胞嘧啶甲基化的化学物质。

<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

胞嘧啶甲基化在脊椎动物物种中高度保守，并且作为表观遗传编程和染色质状态的关键驱动因素，在早期胚胎发育中起着关键作用。酶修饰驱动胞嘧啶的活性甲基化和去甲基化为5-甲基胞嘧啶（5-mC），随后将5-mC氧化为5-羟甲基胞嘧啶、5-甲酰胞嘧啶和5-羧基胞嘧啶。表观遗传重编程是 子宫发育 过程中的关键时期，母体接触化学物质有可能重新编程后代中的表观基因组。这可能会导致不良结局，例如直接表型后果、对成人疾病易感性的长期影响以及遗传表观遗传标记的跨代影响。尽管基于亚硫酸氢盐的测序使研究人员能够以碱基对分辨率询问胞嘧啶甲基化，但基于测序的方法成本过高，因此，无法监测胞嘧啶甲基化跨发育阶段、每种化学物质的多种浓度以及每次治疗复制胚胎的能力。由于自动化 体内 成像、遗传操作、快速宫 外 发育时间和胚胎发生过程中的饲养易于，斑马鱼胚胎继续被用作生理上完整的模型，用于揭示异生素介导的途径，这些途径在早期胚胎发育期间导致不良结果。因此，使用市售的5-mC特异性抗体，我们描述了一种具有成本效益的策略，通过利用全卡口免疫组织化学，自动高内涵成像和统计分析前使用编程语言的高效数据处理，对单个完整斑马鱼胚胎中的胞嘧啶甲基化进行快速有效的时空监测。就目前所知，该方法是第一个在早期发育过程中成功检测和定量斑马鱼 胚胎内 5-mC水平的方法。该方法能够检测细胞团内的DNA甲基化，并且还能够在母体到合子过渡期间检测卵黄定位母体mRNA的胞嘧啶甲基化。总体而言，该方法将有助于快速鉴定在表观遗传重编程过程中有可能原 位 破坏胞嘧啶甲基化的化学物质。

该协议的总体目的是通过评估固定和标记斑马鱼胚胎中荧光的总面积和相对强度来确定处理是否影响5-mC的相对丰度。完成方案后，可以使用荧光体视显微镜首先确定整个安装IHC是否成功。当在FITC或GFP过滤器下观察标记的胚胎时，阳性结果由胚胎内的正FITC信号指示，而阴性结果由对照胚胎内没有荧光表示。使用高内涵筛选系统，这些结果也可以在使用FITC滤光片的图像采集过程中得到确认。此外，...

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

本文描述了一种快速有效地对完整斑马鱼胚胎内正常和异常胞嘧啶甲基化进行时空监测的方案。

快速有效地监测完整斑马鱼胚胎中正常和异常胞嘧啶甲基化的时空监测

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

该协议提供了一种相对快速且具有成本效益的方法，以及自动采集和分析，从而产生5-甲基胞嘧啶的低灵敏度筛选，这是一种常见的表观遗传生物标志物。该技术的主要优点在于它提供了实验设计的灵活性，用户可以根据其模型和开发阶段进行优化。该技术可用于检测化学暴露如何改变发育中生物体内5-甲基胞嘧啶的相对丰度，这通常只能通过昂贵的高trpA方法（如测序）来完成。该方法对于任何旨在利用NCG方法了解5-甲基胞嘧啶响应环境刺激的表观遗传改变的研究都是有用的。首先在收集的早晨准备新鲜的暴露溶液。将五毫升无颗粒系统水加入 60 毫米玻璃培养皿中。然后使用10微升微毛细管玻璃移液器将10微升二甲基亚砜或化学储备溶液转移到系统水中。旋转玻璃培养皿以确保储备溶液消散。向玻璃培养皿中再加入五毫升系统水，并旋转培养皿以使暴露溶液均质化。用玻璃盖盖住玻璃盘，以尽量减少蒸发。接下来，使用充满RO水的喷瓶将胚胎从鱼网转移到100毫米的培养皿中。在受精后 50 小时随机分选 0.75 个活胚胎并去除多余的 RO 水。暴露持续时间结束后，从培养箱中取出胚胎。将所有活胚胎转移到1.5毫升微量离心管中。吸出残留暴露溶液而不吸出任何胚胎。加入500微升冷藏的4%多聚甲醛，并通过倒置几次混合胚胎。让胚胎固定并在 4 摄氏度下将 4% 多聚甲醛过夜。固定胚胎后吸出4%多聚甲醛，将胚胎重新花费在1X PBS中，并通过轻轻移液混合30秒。使用玻璃移液管，小心地将固定胚胎转移到充满RO水的玻璃培养皿中。轻轻旋转 30 秒，然后重复洗涤一次。在设置为五倍放大倍率的体视显微镜下，使用注射器针头对胚胎进行刻膜处理。使用针尖刺穿绒毛膜，轻轻地将其从蛋黄和细胞块上剥离。胚胎脱模后，使用玻璃微毛细管移液器将多达25个完整的胚胎转移到一个免疫组织化学篮中。将IHC篮放入24孔板中。然后从每口井中吸出水。将胚胎重悬于每孔500微升的封闭缓冲液中，用副形和铝箔包裹板以保护其免受光照。将板在 4 摄氏度下以 100 rpm 的速度在轨道振荡器上孵育四小时。孵育后，从每个孔中吸出封闭缓冲液。用500微升一抗溶液代替。请记住用封闭缓冲液孵育一部分载体对照胚胎，以考虑背景噪音。重新包裹板和副成型和铝箔，让板在轨道摇床上在四摄氏度下孵育过夜。第二天从每个孔中取出一抗溶液，并用1x PBST替换。在轨道摇床上用 1x PBST 洗涤每个孔三次，每次洗涤 5 分钟。从每个孔中吸出1x PBST，并将IHC篮子放入装有RO水的玻璃培养皿中。在标准体视显微镜下，将完整的胚胎随机分类到96孔板中，确保每孔一个胚胎。然后从单个井中取出所有RO水，并加入200微升1x PBS。在一个G.Image下用两倍物镜在透射光和FITC下离心板三分钟。选择自动对焦以确保相机的焦点位于板的底部并偏移底部厚度。选择曝光持续时间为 100 毫秒的 FITC 波长，并将自动对焦设置为具有 Z 偏移的激光。在开始板采集之前，观察并确认正确的图像采集和几个孔。图像采集后，使用高内涵筛选系统和定制的自动图像分析程序进行数据分析。选择96孔板上的每个胚胎，以分析荧光的总面积和综合强度。然后将数据点制成表格，并通过“测量”并选择“打开数据日志”将数据导出到电子表格，从而将数据从高内涵筛选系统中导出。将导出的电子表格上传到部署程序包，以对每个孔的数据进行排序和汇总，并按孔数进行筛选。然后使用正确的 Excel 包将汇总的数据导出到电子表格中。在受精后6小时评估斑马鱼胚胎的5-甲基胞嘧啶特异性总面积的分布和综合强度。x轴显示DMSO作为载体或TDCIPP作为阳性对照的暴露。Y 轴表示相对荧光。阈值与载体处理的胚胎显着不同，如胚胎内检测到的5-甲基胞嘧啶的总面积和同一区域内的积分强度所示，表明最大信噪比。保持轭和细胞团的完整性对于正确的IHC检测非常重要。这种方法相对不敏感，仅提供相对丰度的RFU数据。如果需要量化，可以采取更有针对性的方法。

Research

JoVE Journal

Developmental Biology

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

科研

发育生物学

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...