本研究使用的C. 线虫菌株取自秀丽遗传学中心, 由国立卫生研究院 (P40 OD010440) 提供支持。这项工作得到了 NIH 的支持: 1R03AG048578-01, 纽约市立大学-CCRG 1501 至俊良, 和 PSC-纽约市立大学 66184-00 44 和 67248-00 45 至俊良。我们感谢凯西野蛮-邓恩亲切地分享她的实验室空间。所有荧光图像都收集在皇后学院的核心设施。我们感谢威廉 j. 赖斯对手稿的评论。

1. 解决方案
<ol><li>
NGM 板
	<ol>
<li>在2升锥形烧瓶中, 加入3克氯化钠、17克琼脂、2.5 克蛋白胨和975毫升的 dH2o 盖上铝箔的烧瓶口。将烧瓶蒸压50分钟, 冷却20–30分钟。</li>
		<li>然后添加灭菌的解决方案: 1 毫升的1米 CaCl2, 1 毫升5毫克/毫升胆固醇在乙醇, 1 毫升1米 MgSO4, 25 毫升1米 KPO4 (pH 6.0) 缓冲。漩涡的解决方案, 以混合良好。</li>
		<li>使用液体分配器, 将 NGM 溶液分配给60毫米的培养板。把2/3 装满琼脂的盘子填满。将它们存放在4摄氏度的气密容器中, 供将来使用。</li>
	</ol>
</li>
	<li>
1 M KPO4缓冲 (pH 6.0)
	<ol>
<li>在 dH2O 中溶解10.83 克的2PO4和3.56 克的 K4HPO2 , 然后将总容积调整为100毫升。蒸压釜解决方案为15分钟。</li>
	</ol>
</li>
	<li>
M9
	<ol>
<li>溶解3克的2PO4, 6 克的 Na2HPO4, 和 5 g 的氯化钠在 dH2O, 添加1毫升1米 MgSO4, 并调整总容积为1升。</li>
	</ol>
</li>
	<li>
10µg/毫升 DAPI
	<ol>
<li>添加1µL 10 毫克/毫升 DAPI 到999µL 的 dH2O。</li>
	</ol>
</li>
	<li>
95% 酒精 (v/v)
	<ol>
<li>测量95毫升100% 酒精和增加蒸馏水调整容量到100毫升。</li>
	</ol>
</li>
	<li>
30% 丙酮 (v/v)
	<ol>
<li>将30毫升丙酮添加到 dH2O, 并将总容积调整为100毫升。</li>
	</ol>
</li>
</ol>2. 热应力
<ol><li>用目标基因9,10的平移结构生成染色体外阵列线。或者, 从秀丽遗传学中心 (CGC) 获得这样的线, 这是一个公共资源的线虫研究, 或从以前发表的研究实验室。</li>
	<li>通过将蠕虫暴露到 3800 Rad γ辐射9, 将染色体外线整合到基因组中。然后, 选择稳定表达的线条, 使每个动物表达一个标记蛋白, 如 GFP 或滚筒。</li>
	<li>为了消除辐射过程中产生的突变, outcross 至少2x。利用这一转基因线检测在压力下蛋白质表达模式的变化。</li>
	<li>如步骤1.1 所述, 准备 NGM 蠕虫板材。OP50 克隆和培养的细菌在液体 LB 汤过夜。用液体 OP50 培养种子 NGM 板, 在一夜之间孵化37摄氏度孵化皿中的盘子, 种植细菌草坪作为蠕虫的食物来源。在使用前, 在室温下冷却30分钟以上的板材。</li>
	<li>在热应激试验前至少2代, 在20摄氏度不挨饿的情况下种植转基因线。</li>
	<li>选择8–10年轻的妊娠成人 (子宫充满鸡蛋) 到一个新的蠕虫板块, 然后让他们产卵约 3–5 h 和删除所有的成年人从盘子里。</li>
	<li>为了同步蠕虫, 让卵孵化和生长幼虫大约48小时到第四幼虫 (L4) 阶段。检查他们的外阴结构 (半月结构), 以确定 L4 阶段 (图 1, 箭头)11。</li>
	<li>将蠕虫板块与 L4 幼虫放在一个35摄氏度孵化箱中, 2–5 h 以达到热应力。将温度计放在孵化器中, 准确测量温度。</li>
</ol>3. DAPI 染色
<ol><li>
乙醇固定
	<ol>
<li>在玻璃滑梯的中心添加10µL 的 M9 缓冲器。</li>
		<li>从步骤2.8 中选取热休克的蠕虫, 并将它们放入玻璃滑梯上的 M9 缓冲区中。<ol>
<li>或者, 用 M9 把盘子洗掉, 在室温下将其离心 1000 x克1 分钟, 然后丢弃上清。将蠕虫添加到玻璃幻灯片中。</li>
		</ol>
</li>
		<li>用软组织排出多余的液体。在解剖显微镜下观察这一步。</li>
		<li>在蠕虫上添加10µL 95% 的酒精, 让它们风干。观察解剖显微镜下的过程, 不要让动物变得太干燥。乙醇从动物体内蒸发后立即进行下一步。 注: 乙醇蒸发很快。</li>
		<li>重复步骤3.1.4 两次, 以便修复蠕虫。 注意: 这是正常的动物看起来更暗和扭曲。</li>
		<li>添加10µL 的 DAPI (10 µg/毫升) 的蠕虫。立即用盖玻片盖上, 用透明指甲油凝胶封住幻灯片。</li>
		<li>在荧光显微镜下观察10分钟左右的动物。</li>
	</ol>
</li>
	<li>
丙酮固定 (另一种方法)
	<ol>
<li>用 M9 从步骤2.8 中冲洗热震板, 在室温下将其离心 1000 x克, 并丢弃上清液。用1毫升 dH2O 清洗颗粒, 收集颗粒, 并丢弃上清液。</li>
		<li>在室温下添加400µL 30% 丙酮, 15 分钟离心机, 常温下1分钟, 并丢弃上清液。使用500µL 的 dH2O 洗涤动物2x。放弃上清。</li>
		<li>添加200µL 的 DAPI (10 µg/毫升), 或4x 的总量蠕虫的体积, 在管15分钟。</li>
		<li>离心机在 1000 x g 1 分钟, 并丢弃上清。用500µL 洗净颗粒 2x2o 将蠕虫放在玻璃滑梯上, 用盖玻片覆盖, 用透明指甲油凝胶封住幻灯片, 并在荧光显微镜下观察动物。</li>
	</ol>
</li>
</ol>4. 显微成像
<ol><li>打开紫外线光源和荧光显微镜。打开连接到显微镜的计算机。</li>
	<li>在荧光显微镜上加载幻灯片。使用低功耗物镜 (10X) 定位蠕虫, 增加放大功率到 400X, 并专注于标本。</li>
	<li>打开显微镜提供的软件。点击菜单中的 "获取";点击 "相机", 选择相机连接到显微镜;这允许选定的照相机与软件通信。 注: 不同的显微镜在操作上可能有所不同。</li>
	<li>在相同的 "获取" 下, 单击 "多维获取"。这将打开一个新的 "多维获取" 导航窗口。单击 "多声道" 按钮, 所有可用的频道都会出现。</li>
	<li>选择蓝色、绿色和 DIC (差异干扰对比度) 观察标本。在 "多维采集" 导航窗口中, 留下 "蓝 DAPI"、"绿 GFP"、"灰 DIC"、"Nomarski" 频道;右键单击其他通道, 如 "红色罗丹明" 和 "白光场", 以关闭它们。</li>
	<li>
首先, 测量每个通道的曝光时间:
	<ol>
<li>左键点击 "蓝 DAPI" 频道选择它, 然后点击 "测量", 以采取在 DAPI 通道下的实时图像与自动曝光时间。将出现一个具有实时图像的新窗口。</li>
		<li>在 "实时图像" 窗口的左侧, 如果需要, 手动调整曝光时间以使图像达到预期效果。</li>
		<li>最后, 在 "实时图像" 窗口中单击"确定"。这将设置 DAPI 通道下的曝光时间。</li>
	</ol>
</li>
	<li>重复步骤 4.6, 为其他渠道, "绿色 GFP" 和 "灰色 DIC", "Nomarski", 设置的曝光时间, 这些渠道。</li>
	<li>在 "多维采集" 导航窗口中, 单击 "开始" 以根据设定的特定曝光时间自动收集3不同通道下的3幅图像 (见上文)。</li>
	<li>要保存文件, 请转到主菜单, 单击 "文件" 然后在 "另存为"。输入文件名和文件夹名称。将文件保存为默认文件格式, 以保留详细的图像信息以供将来参考。</li>
	<li>
以其他格式导出文件:
	<ol>
<li>转到 "文件", 然后单击 "导出"。这将打开 "导出设置" 窗口。</li>
		<li>设置文件名和文件夹。检查 "创建项目文件夹" 以更好地组织数据。该软件还允许用户指定其他4选项: "生成合并图像"、"使用通道图像的颜色"、"使用通道名称" 和 "仅合并图像"。</li>
		<li>从下拉菜单中选择所需的文件类型, 如 "TIF"、"BMP"、"PSD" 或 "JPG" 等类型。然后单击 "开始" 导出。</li>
	</ol>
</li>
</ol>

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J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

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本文提出了一种快速有效的方法来验证蛋白质亚细胞变化从细胞质到细胞核。蛋白表达由 GFP 融合显示, 而核结构由 DAPI 染色验证 (图 3)。由于染色线虫蛋白具有挑战性, 大多数C. 线虫蛋白亚细胞定位的特点是标记它们与标记蛋白, 如 GFP, 拉扎, mCherry, 和其他18,19,20,21. ...

蛋白质亚细胞定位的变化是反应热应激、饥饿、氧化应激、细胞凋亡、蛋白质磷酸化等内部或外部信号的一种常见机制。例如, 热应力诱导一个 foxo 蛋白成员 DAF-16 核易位1,2和亲凋亡 BCL-2 蛋白出价 translocates 的线粒体后, 接受死亡信号3,4。可以使用各种技术来检测这些更改。西方印迹和生化分离亚细胞结构 (如线粒体或细胞核) 的组合可以很好地实现目标3。然而, 它需要一个特定的抗体对抗蛋白质的兴趣。因此, 一个完善的抗体成为成功的关键。另一种方法是标签不同的亚细胞结构或细胞器与各种标记, 如绿色荧光蛋白 (GFP), 红色荧光蛋白 (RFP), 黄色荧光蛋白 (YFP), 和 mCherry, 同时标签的蛋白质与其他标记的兴趣。然后, 观察他们在共聚焦显微镜下, 以本地化目标5,6。放射性同位素是标记靶蛋白, 然后检测其亚细胞定位7的替代选择。但是, 这种方法需要对放射性废料进行适当的训练和处理。在诸如缺乏特定抗体、缺少适当的标记或 scarceness 等设备的情况下, 需要考虑一种替代方法。为了确定蛋白质核易位, 它是有吸引力的仅标记目标蛋白质与标记和染色核与化学试剂 4 ', 6-diamidino-2-苯基吲哚 (DAPI), 因为这只需要常规荧光显微镜。
Immunolabeling线虫具有抗体是挑战, 由于低渗透性的蛋壳或胶原角质层周围的动物。同时, 由于线虫蛋白与脊椎动物的 orthologs 有明显的差异, 一些商业公司提供特定产品的c. 线虫。小实验室很难为自己生成线虫抗体。社区的研究人员经常使用标记蛋白作为标记来显示蛋白质定位或基因表达。本文以 EXL-1::GFP 为例, 在热应力作用下跟踪蛋白质核易位8。将一个集成的平移exl-1::gfp到动物基因组中, 用于稳定地表达gfp融合的基因。研究表明, exl-1在肠道、体壁肌和其他亚细胞结构中表达8。该协议演示如何将蠕虫同步到第四幼虫 (L4) 阶段, 进行热应激实验, 并在常规荧光显微镜下进行 DAPI 染色、乙醇固定、丙酮固定和成像。

<table><tbody><tr><td>DAPI</td><td>Biotium</td><td>40043</td><td>both Hoechst 33258 and Hoechst 33342 also works well</td></tr><tr><td>OP50</td><td>CGC</td><td>OP50</td><td>https://cgc.umn.edu/</td></tr><tr><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td><td>https://cgc.umn.edu/</td></tr><tr><td>Kim Wipe</td><td>Kimberly-Clark Corporation</td><td></td><td>soft tissue</td></tr><tr><td>Zeiss&amp;nbsp; AX10</td><td>Zeiss</td><td></td><td>fluorescence microscope</td></tr><tr><td>Axiovision Rel 4.8</td><td>Zeiss</td><td></td><td>microscope software</td></tr><tr><td>AxioCam MR Rev3</td><td>Zeiss</td><td></td><td>digital camera</td></tr><tr><td>Incubator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td></td><td></td><td></td></tr><tr><td>Transparent nail polish gel</td><td></td><td></td><td></td></tr><tr><td>60 mm petri dishes</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips</td><td></td><td></td><td></td></tr><tr><td>1-20 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>20-200 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>1-200 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Platinum wire worm pick</td><td></td><td></td><td></td></tr></tbody></table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

本协议采用绿色荧光蛋白 (GFP) 融合蛋白和 4 '、6-diamidino-2-苯基吲哚 (DAPI) 染色法跟踪蛋白质亚细胞定位变化;特别是在热应力条件下的核易位。蛋白质的反应与外部和内部信号相应。一种常见的机制是改变其亚细胞定位。本文介绍了一种跟踪蛋白质定位的协议, 不需要抗体, 放射性标签, 或共焦显微镜。在这篇文章中, GFP 是用来标记的目标蛋白 EXL-1 在C. 线虫, 一个成员的氯离子胞内通道蛋白 (CLICs) 家族, 包括哺乳动物 CLIC4。采用转化与γ辐射相结合的法, 建立了一种集成的转化exl-1::gfp转基因线 (具有启动子和全基因序列), 并稳定地表达了该基因和gfp。最近的研究表明, 在热应力, 而不是氧化应激, EXL-1::GFP 积累在细胞核。将 GFP 信号与细胞核结构和 DAPI 信号重叠, 证实了 EXL-1 亚细胞在应力作用下的定位变化。本协议提出两种不同的固定方法 DAPI 染色: 乙醇固定和丙酮固定。本文提出的 DAPI 染色协议具有快速、高效的功效, 同时保留了 GFP 信号和蛋白质亚细胞定位的变化。此方法只需要使用 Nomarski、FITC 过滤器和 DAPI 过滤器的荧光显微镜。适用于小型实验室设置、本科生研究、高中学生研究、生物技术教室。

氯离子胞内通道蛋白 (点击) 是一种在12种高度保守的多功能蛋白质。许多研究表明, CLICs 调节细胞的压力, 自噬, 凋亡, 癌变, 血管生成和巨噬细胞先天免疫反应在哺乳动物系统13,14,15,16 ,17。线虫中有两个点击同系物: exl-1...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

该协议演示了如何在热应激下跟踪蛋白质核易位, 方法是使用绿色荧光蛋白 (GFP) 融合蛋白作为标记, 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 染色。DAPI 染色协议快速, 保留了 GFP 和蛋白质亚细胞定位信号。

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

detecting-protein-subcellular-localization-green-fluorescence-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

9.5K 次观看.  Borough of Manhattan Community College/CUNY. 该协议演示了如何在热应激下跟踪蛋白质核易位, 方法是使用绿色荧光蛋白 (GFP) 融合蛋白作为标记, 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 染色。DAPI 染色协议快速, 保留了 GFP 和蛋白质亚细胞定位信号。

视频: Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

科研

发育生物学

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

秀丽隐杆线虫 用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. ...

遗传学

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

免疫与感染

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

定量 秀丽隐杆线 虫和肝癌细胞系中的线粒体自噬

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

作者聚焦：使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

使用细胞器特异性染料检测 秀丽隐杆 线虫和哺乳动物细胞中的线粒体自噬

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

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C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

秀丽隐杆线虫用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

成像 C。线虫胚胎使用啶显微镜和开放源码软件

时间推移显微镜早期胚胎发育线虫

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

方法来评估肌肉的亚细胞中

自动化秀丽隐杆线虫的聚集定量

使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

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C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

秀丽隐杆线虫 用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

成像 C。线虫胚胎使用啶显微镜和开放源码软件

时间推移显微镜早期胚胎发育线虫

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

方法来评估肌肉的亚细胞中

自动化秀丽隐杆线虫的聚集定量

使用细胞器特异性染料检测 秀丽隐杆 线虫和哺乳动物细胞中的线粒体自噬

秀丽隐杆线虫用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬