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

这项工作得到了中国自然科学基金会（资助编号：81870239、81741005、81972999）和江苏省高等学校优先学术项目发展的支持。

本研究中使用的野生斑马鱼（AB）是从中国武汉国家斑马鱼资源中心获得的。这里概述的所有动物程序都经过了苏乔大学伦理委员会动物护理研究所的审查和批准。
1. 下午2.5 采样和有机化合物提取
注：下午2.5 收集于中国苏州市区，2015年8月1-7日，如前5日所述。
<ol><li>在 500 °C 消声炉中烘烤 47 mm 石英膜过滤器 2 小时，?...

<ol>
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虽然斑马鱼是研究环境化学物质心脏发育毒性的极好的脊椎动物模型，但由于胚胎心脏体积小，很难获得足够的蛋白质进行西方污点分析。因此，我们提出了一种敏感的免疫荧光方法，用于量化暴露在PM2.5下的斑马鱼胚胎心脏中DNA损伤生物标志物的蛋白质表达水平。
在解剖过程中，保持心脏的完整性是很重要的。根据我们的经验，在 72 hpf 下执行隔离相对容易。此外?...

空气污染是目前世界面临的一个严重的环境问题。环境细颗粒物（PM2.5）是空气质量最重要的指标之一，可携带大量有害物质进入血液循环系统，对人体健康造成严重危害。流行病学研究表明，PM2.5暴露可导致先天性心脏缺陷（CHDs）2，3的风险增加。动物实验的证据表明，PM2.5可导致斑马鱼胚胎和小鼠后代心脏发育异常，但PM2.5心脏发育毒性的分子机制仍鲜为人知。
DNA损伤可引起细胞周期骤停并诱发凋亡，从而可能广泛破坏祖细胞的潜力，进而损害心脏发育。有充分证据表明，环境污染物，包括PM2.5，有可能通过氧化应激机制8，9攻击DNA。人类和斑马鱼心脏发育都对氧化应激10、11、12敏感。8-OHdG 是一种氧化性 DNA 损伤标记，+H2AX 信号是 DNA 双链断裂的标志。N-乙酰-L-半胱氨酸（NAC）是细胞内半胱氨酸和谷胱甘肽的合成前体，被广泛用作抗氧化化合物。在这项研究中，我们使用NAC来研究氧化应激在下午2.5诱发DNA损伤13中的作用。
斑马鱼作为脊椎动物的模型，已被广泛用于研究心脏发育和人类心血管疾病，因为心脏发育机制在脊椎动物中保存高度。使用斑马鱼作为模型的优势包括它们体积小、繁殖能力强和饲养成本低。这些研究特别感兴趣的是，斑马鱼胚胎在早期发育过程中不依赖于循环系统，并且能够存活下来，严重心脏畸形14。此外，它们的透明度允许在显微镜下直接观察整个身体。因此，斑马鱼胚胎提供了一个绝佳的机会，以评估参与诱导心脏发育毒性的分子机制，由于接触各种环境化学品5，16，17。我们之前曾报道，PM2.5引起的氧化应激导致DNA损伤和凋亡，导致斑马鱼18的心脏畸形。在这项研究中，我们提供了一个详细的协议，用于调查PM2.5引起的斑马鱼胚胎心脏DNA损伤。

<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

环境细颗粒物（PM2.5）暴露可导致心脏发育毒性，但潜在的分子机制尚不清楚。8-羟基-2'脱氧酶（8-OHdG）是氧化DNA损伤的标记，+H2AX是DNA双链断裂的敏感标记。在这项研究中，我们旨在通过免疫荧光检测斑马鱼胚胎心脏的PM2.5诱导8-OHDG和+H2AX变化。斑马鱼胚胎在受精后2小时（hpf）2小时时，以5微克/mL的PM2.5 处理可提取的有机物质（EOM）。DMSO 被用作车辆控制。在72马力，心脏被解剖从胚胎使用注射器针和固定和渗透。被阻塞后，用原样抗体对8-OHdG和+H2AX进行探测。然后用二次抗体清洗和孵育样品。由此产生的图像在荧光显微镜下观察，并使用 ImageJ 进行量化。结果表明，EOM从下午2.5 显著增强8-OHDG和+H2AX信号在斑马鱼胚胎的心脏。然而，NAC作为活性氧物种（ROS）的清除剂，部分抵消了EOM引起的DNA损伤。在这里，我们提出了一个免疫荧光方案，用于研究DNA损伤在PM2.5诱发的心脏缺陷中的作用，可用于检测斑马鱼胚胎心脏中环境化学诱发的蛋白质表达变化。

这种免疫荧光检测是测量暴露于环境化学物质的斑马鱼胚胎心脏蛋白质表达变化的一种敏感而具体的方法。
在这个具有代表性的分析中，在没有抗氧化剂NAC的情况下暴露于PM2.5的胚胎被评估为存在心脏畸形（图1）。观察到，与 DMSO 控制治疗的心脏相比，PM2.5导致心脏畸形发生显著增加，?...

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

该协议使用免疫荧光检测检测斑马鱼胚胎解剖心脏中PM2.5诱发的DNA损伤。

使用免疫荧光检测斑马鱼胚胎心脏PM2.5诱发DNA损伤

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

这种方法可用于准确、轻松地检测斑马鱼心脏中目标蛋白的表达。目标蛋白的变化通过测量免疫荧光直接检测。执行斑马鱼胚胎收集和处理，如文本手稿中所述。受精后72小时，将胚胎转移到玻璃滑梯上，并在立体显微镜下观察。记录心脏畸形，如腹膜水肿、循环改变和尺寸缩小。使用单向 ANOVA 计算畸形率并分析组之间的差异，然后是土耳其的多次比较测试。在用每毫升MS222000克麻醉胚胎后，记录心跳30秒，并使用图像J软件量化心率。在立体显微镜下用一次性注射器针仔细剖析斑马鱼胚胎中的心脏。使用疏水屏障笔在干净的玻璃滑梯上画一个圆圈。将50微升4%的甲醛加入PBS的1.25微升，形成固定溶液，然后将三颗解剖的心放入一个疏水屏障圈中，在室温下孵育20分钟。在显微镜下解除溶液，并在室温下干燥样品至少五分钟。每次洗涤三次 PBST 的幻灯片，每次洗涤五分钟。将 50 微升 BSA 添加到 PBST 的 1000 微升中，以获得 5% 的 BSA 溶液，并将滑梯在潮湿的腔室中孵育一小时，以阻止非特定抗体结合。将溶液除以每次洗涤三次，每次用 PBS 清洗五分钟。在PBST的296微升中稀释两种小鼠单克隆抗体的微光素，对8-羟基二氧观氨酸和两种针对伽马-H2AX的兔子多克隆抗体微升，以获得有效的初级抗体鸡尾酒溶液。在室温下或在四摄氏度的夜间，用50微升原抗体鸡尾酒溶液在潮湿的室内孵化心脏样本至少一小时。将溶液除以每洗五分钟，然后用 PBST 清洗样品三次。在PBST的500微升中稀释一个FITC标签山羊抗鼠二级抗体的微升和一个微升的psy-3山羊抗兔二级抗体，以获得有效的二级抗体鸡尾酒溶液。在黑暗中室温下，用二次抗体孵育样品一小时。将溶液除以每次洗涤三次，每次用 PBS 清洗五分钟。在样品中加入20微升DAPI用于核染色，并在室温下孵育30分钟。将盖片涂在滑梯上，用指甲油密封，以防止干燥和移动。在荧光显微镜下对样品进行成像，并使用 ImageJ 软件量化心脏区域的荧光信号。使用 DMSO 控制样本的平均值计算相关变化，并确定数据的统计意义。在没有抗氧化剂N-乙酰-L-半胱氨酸或NAC的情况下暴露于PM2.5的胚胎，被评估为存在心脏畸形。与 DMSO 控制治疗的心脏相比，从 PM 2.5 开始的 EOM 成本显著增加心脏畸形，如心周水肿、改变循环和缩小大小。暴露于 EOM 的胚胎的心率也显著下降。增加 NAC 衰减 EOM 诱发的心脏缺陷。免疫荧光检测用于评估 EOM 处理组织中的 DNA 损伤程度。用EOM治疗的斑马鱼胚胎的心脏中，8-羟基二氧观光素和伽马-H2AX表达水平显著增加，表明氧化DNA损伤和DNA双链断裂分别增加。EOM 引起的 DNA 损伤部分抵消了 NAC 补充。在尝试此协议时，请小心用 PBS 清洗样品，因为斑马鱼心脏可能会从滑梯上剥落。

Research

JoVE Journal

Developmental Biology

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

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Microbead Implantation in the Zebrafish Embryo

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...