这项工作是有可能的 NIH 补助金: R01GM109028 (爱护动物协会)

1. 尼罗河红 (NR) 脂质染色
<ol><li>5毫克/毫升 NR 溶液的制备<ol>
<li>在一个500毫升瓶中, 加入100毫克的 NR 粉到200毫升100% 丙酮。</li>
		<li>用铝箔盖住瓶子, 以免暴露在光线下。</li>
		<li>使用前, 在黑暗中搅动2小时的溶液。</li>
		<li>长期使用, 将 NR 库存解决方案储存在密封的瓶子里, 不暴露任何光线。根据研究需要, 规模化 NR 库存解决方案。确保在 NR 染色实验中使用相同的库存解决方案, 以获得一致的染色和成像结果。</li>
	</ol>
</li>
	<li>NR 工作液的制备<ol>
<li>每1毫升40% 异丙醇 (v/v), 添加6µL 的 NR 库存解决方案。</li>
		<li>为每个样品准备600µL 的 NR 工作溶液。 注: 在染色前, 应先制作新鲜的 NR 工作溶液。根据 NR 库存解决方案的需要, 使用15毫升或50毫升毕业圆锥管。</li>
	</ol>
</li>
	<li>NR 脂质染色蠕虫的制备<ol>
<li>生长蠕虫到早 L4 阶段在20°c 在线虫生长培养基 (NGM) 播种与晚日志 OP50大肠杆菌。</li>
		<li>用1毫升的1x 磷酸盐缓冲盐水 + 0.01% X-100 (PBST) 溶液把盘子上的蠕虫洗净, 并将蠕虫悬浮在1.5 毫升离心管中。</li>
		<li>将蠕虫在 560 x g上离心1分钟. 移除上清, 并重复此步骤, 直到大肠杆菌从悬浮中清除。</li>
		<li>加入100µL 40% 异丙醇的蠕虫颗粒, 并在室温下孵化3分钟。</li>
		<li>将蠕虫在 560 x g上离心1分钟, 然后移除上清, 而不破坏蠕虫颗粒。 注: 使用较大的 PBST, 如果使用更大的盘子或染色更多的蠕虫每板, 从盘子里清洗蠕虫。在样品固定前不要在 PBST 中清洗15分钟以上的蠕虫。如果清液未清除细菌, 则进行额外洗涤。使用 nutator 或摇杆进行40% 异丙醇的孵化。始终监控管道, 以确保充分的样品搅拌正在发生。</li>
	</ol>
</li>
	<li>脂质染色与 NR<ol>
<li>在黑暗中, 为每个样品添加600µL 的 NR 工作溶液。反转三次管, 完全混合在 NR 溶液中的蠕虫。</li>
		<li>旋转样品在黑暗中在室温下2小时。</li>
		<li>孵化后, 将蠕虫在 560 x g上离心1分钟, 然后移除上清。</li>
		<li>添加600µL 的 PBST 和孵化的样品在黑暗中30分钟, 以去除过量的 NR 染色。</li>
		<li>离心样品在 560 x g为1分钟并且去除所有, 但大约50µL 上清。 注: NR 孵化不需要搅拌。在这个步骤中, 蠕虫的解决是常见的。</li>
	</ol>
</li>
	<li>显微镜成像用幻灯片的制备<ol>
<li>在剩余的上清并用重悬蠕虫颗粒。</li>
		<li>将5µL 的蠕虫悬浮在显微镜下滑动, 并小心地放置盖玻片, 以避免捕捉气泡。</li>
		<li>在成像蠕虫之前用指甲油封住盖玻片。</li>
		<li>一次只准备几张幻灯片。这将确保 NR 成像在样本中保持不变。在6小时后, 对 nr 染色图像质量的影响降低了, 不容易观察到离散的脂滴, 背景荧光增加, 干扰了 nr 信号的检测。</li>
	</ol>
</li>
	<li>NR 染色蠕虫的影像学研究<ol>
<li>图像的蠕虫在5X 放大, 以捕获几个动物的每一个视野。</li>
		<li>切换到10X 放大倍数以更好地量化单个蠕虫。</li>
		<li>使用 FITC/GFP 通道对 NR 染色的蠕虫进行图像检测, 并尝试不同的曝光时间来确定量化的最佳条件。</li>
		<li>以 TIF 格式保存文件, 以避免由于压缩而丢失数据。 注: 典型曝光时间范围从100-1000 毫秒起, 具有最佳曝光时间, 可用于所有样本以保持成像一致性。</li>
	</ol>
</li>
	<li>NR 染色图像量化<ol>
<li>将显微照片上载到 ImageJ。在 "插件" 下拉菜单中, 如果不识别图像文件, 请使用 "生物格式" 函数。</li>
		<li>在 "图像" 下拉菜单中的 "堆栈" 函数下, 在比较多个显微图像时, 使用图像到堆栈来创建映像堆栈。</li>
		<li>在同一下拉菜单中, 调整图像堆栈的亮度/对比度, 并通过单击 "应用" 将更改传播到所有图像。</li>
		<li>利用多边形选择工具对每个图像蠕虫进行描述, 并使用分析下拉菜单中的度量函数来量化 NR 发出的荧光强度。</li>
		<li>对于每个图像, 测量五背景位置和计算平均背景荧光强度。</li>
		<li>使用公式 n = G (A x B) 减去每个成像蠕虫的背景荧光强度, 其中 n 代表净荧光, G 用于总荧光, A 用于全蠕虫区域, B 用于平均背景荧光。</li>
		<li>为了使荧光强度通过蠕虫大小正常化, 将每个蠕虫的净荧光量除以其总面积。结果被报告作为荧光强度, 在任意单位 (天文单位), 每像素。 注意: 避免以 JPEG 格式导出图像。当压缩使文件更易于存储时, 数据完整性会受到这种格式的影响。确保对所有图像进行了相同的修改和分析。请记住, 在不同的放大中, 应包括一个刻度条以正确评估大小。为了更好地可视化使用 ImageJ 的荧光强度的差异, 将图像类型从 RGB 切换到8位, 然后从 "图像查找表" 菜单中选择 "火"。这将创建一个热图图像, 其中增加的颜色亮度与增加的荧光强度相关。</li>
	</ol>
</li>
</ol>2. 油脂红 O 染色 (破产)
<ol><li>库存解决方案的准备<ol>
<li>在一个250毫升的瓶子, 添加500毫克的破产剂粉到100毫升100% 异丙醇和混合它好。</li>
		<li>一旦准备好, 储存的解决方案紧密密封, 没有光暴露。</li>
	</ol>
</li>
	<li>制定破产工作解决方案<ol>
<li>稀释在水中的库存溶液 (3:2) 到60% 异丙醇。</li>
		<li>使用前, 通过0.2 µm 醋酸纤维素无菌注射器过滤器过滤工作的破产解决方案。 注: 为最佳解决方案质量, 前一天准备工作的破产解决方案, 并让它混合过夜。但是, 如果当天需要解决方案, 在使用前至少2小时稀释并混合破产解决方案。在摇动前, 将锥形管包裹在石蜡胶带中, 以避免泄漏的破产解决方案。</li>
	</ol>
</li>
	<li>口腔脂质染色蠕虫的制备<ol>
<li>在线虫生长培养基 (NGM) 上生长20摄氏度的蠕虫, 并将后期记录 OP50大肠杆菌播种到理想的生命期。</li>
		<li>添加1毫升的 PBST 溶液的板和漩涡, 直到所有的蠕虫是关闭板。倾斜的盘子和洗涤它与1毫升的 PBST。将蠕虫悬浮液转移到1.5 毫升离心管。</li>
		<li>将蠕虫在 560 x g上离心处理1分钟. 除去上清液, 不干扰颗粒, 重复洗涤步骤1毫升的 PBST 三次。除去所有上清, 但100µL。</li>
		<li>添加600µL 40% 异丙醇的蠕虫颗粒和岩石在室温下3分钟。</li>
		<li>将蠕虫在三十年代的 560 x g上离心, 除去所有上清, 但100µL, 而不破坏蠕虫颗粒。 注: 如果进行高通量染色, 使用更高的 PBST 来清洗板上的蠕虫。在样品固定前不要在 PBST 中洗15分钟以上的蠕虫。如果清液未清除细菌, 则额外冲洗。使用 nutator 或摇杆进行40% 异丙醇的孵化。始终监测管, 以确保充分的样品搅拌正在发生。</li>
	</ol>
</li>
	<li>脂质染色与破产处理<ol>
<li>为每个样品添加600µL 的工作解决方案。把管子倒置三次, 在破产的时候把虫子混合好。</li>
		<li>在室温下将样品旋转30转2小时。</li>
		<li>离心样品在 560 x g 1 分钟, 并消除所有上清, 但100µL。</li>
		<li>并用重悬的样品在600µL 的 PBST 和旋转管在 30 rpm 30 分钟, 以消除过剩的破产污点。</li>
		<li>离心机样品在 560 x g为1分钟和排除除50µL 的上清。</li>
		<li>为了更好地区分动物的生殖和肠道细胞的位置, 染色核通过增加1µL (2-(4-amidinophenyl)-1 h indole-6-carboxamidine) (DAPI) 每1毫升的破产工作解决方案。在安装幻灯片进行成像前添加 DAPI, 进行2小时的破产检测。肠道细胞核体积较大, 性腺由发育中的生殖细胞区分。 注: 在破产后染色后, 蠕虫可能会附着在离心管的两侧, 不能形成明显的颗粒。如果发生这种情况, 再离心一次蠕虫, 或者允许蠕虫以重力沉降至少10分钟。</li>
	</ol>
</li>
	<li>蜗杆成像用滑动片的制备<ol>
<li>并用重悬在剩余的上清的蠕虫, 并混合解决方案好。</li>
		<li>将5µL 的蠕虫悬浮在显微镜下滑动, 并小心放置盖玻片, 确保没有气泡形成。</li>
		<li>用指甲油和图像蠕虫盖玻片。 注意: 在固定和染色后, 蠕虫可能是非常刚性和困难的吸管。为了规避这一点, 通过切断他们的小费, 使宽孔吸管。</li>
		<li>将幻灯片存储在微离心机架中, 温度为4摄氏度, 如果染色后不立即成像, 则可达24小时。</li>
	</ol>
</li>
	<li>染污蠕虫的影像学研究<ol>
<li>使用具有彩色功能的照相机来图像被污染的蠕虫。</li>
		<li>使用5X 放大倍数在一个视野中图像数个蠕虫。</li>
		<li>切换到10X 放大倍数, 以便更好地检查单个蠕虫。</li>
		<li>以 TIF 格式导出图像, 以避免由于压缩导致的数据丢失。 注: 如果与破产处理 + DAPI 染色, 切换到可捕获荧光的彩色相机。</li>
	</ol>
</li>
	<li>染污蠕虫的图像分析<ol>
<li>将显微图像上传到 ImageJ。在 "插件" 下拉菜单中, 如果不识别图像文件, 请使用 "生物格式" 函数。</li>
		<li>在 "图像" 下拉菜单中的 "堆栈" 函数下, 在比较多个显微图像时, 使用图像到堆栈来创建映像堆栈。</li>
		<li>在同一下拉菜单中调整亮度/对比度, 以提高脂滴的可见度。</li>
		<li>根据整个动物体内或特定组织的脂质积累对图像进行分类。</li>
		<li>为了更好地评价脂质定位和使用 ImageJ 识别非特定图像工件, 请选择图像下拉菜单下的颜色函数, 然后单击堆栈到 rgb 以显示每个 rgb 分量的信号。</li>
	</ol>
</li>
</ol>

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作者声明没有利益冲突。

肥胖和代谢性疾病发病率的上升使线虫成为研究细胞和组织中脂肪积累机制的合适模型。最近的证据表明, 脂质水平的变化与细胞过程相关, 从胰岛素信号8, 激活激素受体2, 到生殖输出5。与无标签显微镜和色谱方法相比, 尼罗河红和油红色 O 是相对便宜的染料, 用于染色蠕虫的中性脂一贯和重现性 10, 12, 13.<sup cla...

血脂对生命至关重要。它们是膜的组成部分, 作为辅助信使和信号传感器, 在储能中具有重要的作用。当脂质代谢是失调, 它导致疾病, 如肥胖和 II 型糖尿病, 这是紧迫的公共卫生问题9。秀丽线虫(线虫) 是研究脂质代谢的优秀模型有机体, 因为它具有相对较短的生命周期、透明的身体、已知的细胞谱系和完全有序的基因组。主要是雌雄同体,线虫允许研究人员在短时间内培养大量的等基因系动物, 以进行高通量的前基因筛, 研究多种代谢基因和通路4。这种方法在273种线虫脂代谢基因中发现了高度的保护, 如人类、老鼠、老鼠和果蝇。此外, 在线虫中有300多种脂质基因有与代谢综合征相关的疾病的人类 orthologues11。传统上, 对线虫中脂质贮存的检查大多依赖于染料标记的化验, 这为脂质积累提供了可靠的信息。不太常见的是关于脂质的本地化和测量不同组织脂质丰度差异的描述。然而, 最近的工作表明, 脂质分布可以和脂质积累一样重要6。
最近, 研究已经开始整合的方法, 如高效液相色谱-质谱 (HPLC), 气相色谱-质谱 (GC), 和相干反斯托克斯拉曼散射 (汽车) 显微镜, 以解决通过直接分析脂质提取物、特定脂质组分和脂质沉积物的含量, 分别为10、11, 以染色为基础的方法的缺点。此外, 汽车显微学已经表明, 尼罗河红色只能作为一个代理的脂肪积累时, 用作固定染料, 因为它作为一个重要的污点, 导致非目标染色的自动荧光器10。然而, 与这些色谱和显微学方法相关的必要技术专长和成本使得它们在许多研究问题上的使用站不住脚。在本文中, 我们讨论了一种方便可靠的方法来锁定和染色中性脂质矿床的C. 线虫使用尼罗河红和油红色 O 区分脂质丰度在整个动物和特定组织。
尼罗河红, 9-二氨基-5 h 苯并 [α] phenoxazine-5-一, 是一种 benzophenoxazone 染料, 容易溶解在各种有机溶剂, 但大多不溶于水。它是一种很好的 lysochrome 染料, 用来染色中性脂类, 如甘油三酯或胆固醇酯, 因为它具有强烈的颜色, solubilizes 良好的脂质, 与周围组织的相互作用微不足道, 而且不溶于溶剂比血脂.它的激发和发射最大值为450-500 和 520 nm, 分别为1。当对尼罗河红色染色的线虫进行绿色荧光观察时, 可以在整个肠道和其他组织中以团簇或均匀分散的方法观测离散脂质体, 这取决于动物的基因型或实验性治疗7。
油红色 O 是一种 lysochrome 脂溶性染料, 用于染色甘油三酯和脂蛋白。它被称为偶氮染料, 因为它的化学结构含有两个偶氮基团附着在三芳香环上。这是很难电离, 使其高度溶于脂质。其着色色为红色, 其光吸收最大值为 518 nm 3。C. 线虫染色的油红色 O 显示红色脂滴, 突出反对动物的透明身体, 这有助于定性评估不同组织之间的脂质分布6。

<table><tbody><tr><td>Imager.M2m Microscope</td><td>Zeiss</td><td>n/a</td><td>Fluorescence microscope</td></tr><tr><td>ERC5s camera</td><td>Axiocam</td><td>n/a</td><td>Color-capable</td></tr><tr><td>MRm camera</td><td>Axiocam</td><td>n/a</td><td>Fluorescence-capable</td></tr><tr><td>Nile red</td><td>Thermo Fisher</td><td>N1142</td><td>Lipid Stain</td></tr><tr><td>Oil red O</td><td>Alfa Aesar</td><td>A12989</td><td>Lipid Stain</td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td>DNA stain</td></tr><tr><td>Isopropyl Alcohol</td><td>BDH</td><td>BDH1133-1LP</td><td>Fixative solution</td></tr><tr><td>0.2 &amp;micro;m seterile syringe filter</td><td>VWR</td><td>28145-477</td><td>Cellulose acetate filter</td></tr><tr><td>Centrifuge 5430</td><td>Eppendorf</td><td>5428000015</td><td>Centrifuge</td></tr><tr><td>Shaker Rotisserie</td><td>Lab Quake</td><td>400110Q</td><td>Shaker</td></tr><tr><td>Tube Rotator</td><td>VWR</td><td>10136-084</td><td>Rotator</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7758-11-4</td><td>NGM</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7778-77-0</td><td>NGM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Alfa Aesar</td><td>7786-30-3</td><td>NGM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>10035-04-8</td><td>NGM</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td>NGM</td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>57-88-5</td><td>NGM</td></tr><tr><td>Peptone</td><td>BD Biosciences</td><td>211677</td><td>NGM</td></tr><tr><td>Agar</td><td>Teknova</td><td>L9110</td><td>NGM</td></tr><tr><td>LB media</td><td>Sigma-Aldrich</td><td>L3147</td><td>Bacterial growth</td></tr></tbody></table>

quantification of lipid abundance and evaluation of lipid distribution in caenorhabditis elegans by nile red and oil red o staining

秀丽线虫是一种特殊的模型有机体, 用于研究脂质代谢和能量稳态。许多脂质基因在人类中被保存, 并与代谢综合征或其他疾病有关。通过固定染料或无标签的方法对机体脂质积累进行检查。像尼罗河红色和油红色 O 的固定污渍是廉价的, 可靠的方法来定量测量脂质水平, 并定性地观察各组织的脂质分布。此外, 这些污渍允许高通量筛选各种脂代谢基因和途径。此外, 其疏水性的性质有利于脂溶性, 减少与周围组织的相互作用, 并防止离解进入溶剂。虽然这些方法是有效的检查一般脂质含量, 他们没有提供详细信息的化学成分和多样性的脂质矿床。为了这些目的, 无标签的方法, 如 GC-MS 和汽车显微术更适合, 他们的成本尽管。

SKN-1 是一个 bZip, 细胞保护转录因子, 与哺乳动物 NRF2 同源, 并已被证明介导脂肪酸氧化。根据其饮食中的葡萄糖浓度, 带有组成性激活的 skn-1 等位基因的蠕虫在沾上尼罗河红色7时显示出不同的血脂水平. 图 1A-c显示已激活的skn-1动物, 它们暴露在导致血脂水平升高的条件下。使用 FITC/GFP 通道捕获的 NR 荧光是...

Watch this Scientific Journal Video about Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining at JoVE.com

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

固定秀丽线虫的尼罗河红色染色是一种定量测量中性脂质矿床的方法, 而油红色 O 染色有助于定性评价组织间脂质的分布。

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

quantification-lipid-abundance-evaluation-lipid-distribution

Research

JoVE Journal

Biology

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

20.1K 次观看.  University of Southern California. 固定秀丽线虫的尼罗河红色染色是一种定量测量中性脂质矿床的方法, 而油红色 O 染色有助于定性评价组织间脂质的分布。

视频: Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

科研

化学

使用 3D 荧光脂滴重建评估肝脂肪变性

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

作者聚焦： 使用 3D 重建分析荧光染色脂滴用于肝脂肪变性评估  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

生物化学

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

生物学

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Worm-align and Worm_CP, Two Open-Source Pipelines for Straightening and Quantification of Fluorescence Image Data Obtained from Caenorhabditis elegans

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This problem is aggravated with increasing density of worms and creates challenges for imaging and quantification. We developed a FIJI-based workflow, Worm-align, that can be used to generate single- or multi-channel montages of user-selected, straightened and aligned worms from raw image data of C. elegans. Worm-align is a simple and user-friendly workflow that does not require prior training of either the user or the analysis algorithm. Montages generated with Worm-align can aid the visual inspection of worms, their classification and representation. In addition, the output of Worm-align can be used for subsequent quantification of fluorescence intensity in single worms, either in FIJI directly, or in other image analysis software platforms. We demonstrate this by importing the Worm-align output into Worm_CP, a pipeline that uses the open-source CellProfiler software. CellProfiler&#8217;s flexibility enables the incorporation of additional modules for high-content screening.&#160;As a practical example, we have used the pipeline on two datasets: the first dataset are images of heat shock reporter worms that express green fluorescent protein (GFP) under the control of the promoter of a heat shock inducible gene hsp-70, and the second dataset are images obtained from fixed worms, stained for fat-stores with a fluorescent dye.

Worm-align and Worm_CP, Two Open-Source Pipelines for Straightening and Quantification of Fluorescence Image Data Obtained from Caenorhabditis elegans

Worm-align/Worm_CP is a simple FIJI/CellProfiler workflow that can be used to straighten and align Caenorhabditis elegans samples and to score whole-worm image-based assays without the need for prior training steps. We have applied Worm-align/Worm_CP to the quantification of&#160;heat-shock induced expression in live animals or lipid droplets&#160;in fixed samples.

蠕虫对齐和Worm_CP，两个开源管道，用于从卡诺哈布迪蒂斯精英获得的荧光图像数据的直化 和定量

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

秀丽隐杆线虫长寿的脂质补充和基因转录分析

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

摘要

探索更多视频

此视频中的章节

一种简单快速的协议用荧光测量中性脂肪在藻细胞

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

生化和高通量的微观评估的脂肪含量

果蝇幼虫的欧诺细胞的解剖和脂滴染色

蠕虫对齐和Worm_CP，两个开源管道，用于从卡诺哈布迪蒂斯精英获得的荧光图像数据的直化和定量

使用刺激拉曼散射显微镜对卡诺哈布迪炎电子人中的脂质存储动力学进行无标签成像

秀丽隐杆线虫长寿的脂质补充和基因转录分析

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

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一种简单快速的协议用荧光测量中性脂肪在藻细胞

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

生化和高通量的微观评估的脂肪含量

果蝇幼虫的欧诺细胞的解剖和脂滴染色

蠕虫对齐和Worm_CP，两个开源管道，用于从卡诺哈布迪蒂斯精英获得的荧光图像数据的直化 和定量

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

秀丽隐杆线虫长寿的脂质补充和基因转录分析

蠕虫对齐和Worm_CP，两个开源管道，用于从卡诺哈布迪蒂斯精英获得的荧光图像数据的直化和定量

使用刺激拉曼散射显微镜对卡诺哈布迪炎电子人中的脂质存储动力学进行无标签成像