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.第一种方法是对总脂质水平进行量化, 第二种方法有助于评价组织中的脂质分布, 如皮下组织、肠道和细菌线。在联合使用时, NR 和破产的研究人员可以确定基因型和环境变化如何影响脂质积累以及蠕虫在哪里发生这些变化。
然而, 这些染料并没有直接评估与汽车显微学12相同的非侵入性的脂质积累。因此, 他们容易在固定和染色过程中出现错误, 这可能会损害测量准确度13。此外, 这些染料可能无意中与脂褐素和肠道颗粒相互作用, 导致荧光无关的细胞内中性脂肪存储10, 14.此外, 在脂质水平较低的情况下, NR 和破产的染色不能区分, 如果结果是由于渗透性问题或减少脂肪积累。此外, 这些污渍对光的敏感性, 需要在储存和积极处理, 限制降解和照片漂白的特殊措施。但是, 如果在执行和绘制基于标签的化验结论时小心谨慎, NR 和破产检测是向前基因筛的优良手段, 研究脂质代谢通路, 并检查它们与其他生理功能.建议使用daf-2和fat-6蠕虫对增加和减少的脂质积累分别进行相对比较。对 NR 和破产的染色的洗涤和采取的步骤是相似的。因此, 这两个可以在同一天执行。但是, 只有带有未经过检测的蠕虫的幻灯片可以存储在以后进行成像, 因为 NR 染色在6小时后是不一致的。在洗涤过程中, 重要的是包括海卫 100-X 不仅提高了对 NR 和破产的渗透率, 而且也避免了由于过度坚持 plasticware 的蠕虫损失。此外, 蠕虫在染色前应清洗少于30分钟, 以确保每个污渍的最大渗透性。虽然 NR 和破产的潜伏期可以减少到1小时和30分钟, 但鼓励遵循本文建议的时间, 以更一致的染色和成像结果。在使用前, 长期暴露 NR 溶液对光和未能过滤破产解决方案将增加成像过程中的背景荧光。执行这些染色协议需要3-4 小时, 除了成像时间。虽然染色孵化提供了最多 2 h 的步骤之间的停顿, 但强烈建议, 这些协议进行尽可能少的中断, 以减少所有的错误来源, 所有的染料基础的脂质量化方法和考试.

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 &#181;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>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>7758-11-4</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>7778-77-0</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Alfa Aesar</td>
			<td>7786-30-3</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>CaCl2</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 荧光是沿肠道突出的, 但在头部、尾部和肠道腔内较暗。随着血脂水平的提高, 分离的 NR 染色颗粒更难以辨别, 这可能需要较低的暴露时间。该方法采用荧光强度作为脂质积累的定量代理, 但不足以区分非脂肪贮存的细胞结构的非特异性染色, 容易引起肠道自动信号干扰。荧光.因此, 替代标签和非标签方法必须并行使用, 以明确量化中性脂在动物的10。
年龄依赖性的身体耗竭脂肪 (日本自卫队), 是一种表型, 发生在衰老蠕虫, 即动物显示减少体细胞脂, 而生殖细胞脂质保持不变的6。油红色 O 染色不是一个可靠地量化脂质水平的方法, 但是很好的可视化脂质定位, 是有用的, 以确定脂肪耗竭表型, 如日本自卫队在蠕虫。虽然根据日本自卫队的存在或不存在对蠕虫进行分类很容易, 但识别中等脂肪损失的动物可能很难(图 2A)。与非日本自卫队动物相比, 它在人体内显示出明亮的红色染色, 半透明区域很少(图 2B), 日本自卫队蠕虫在肠道细胞中表现出明显的脂肪消耗(图 2C)。在开发日本自卫队的过程中, 动物(图 2D) 经常显示出半透明的斑点, 大多数脂肪的损失最终会出现。此外, 日本自卫队蠕虫可能仍然具有明亮的红色染色在头部和尾部地区, 因为表型不是由完整的躯体脂肪损失定义, 而是由广泛的脂肪消耗相对于非老年 (非日本自卫队) 动物。因此, 使用油红色 O 染色, 可以定性地确定脂质的定位和鉴别的表型, 大大改变脂肪沉积的蠕虫。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57352/57352fig1.jpg"> 图 1: 用尼罗河红色染色脂质.尼罗河红色染色的活化的skn-1突变体的条件下, 增加脂积累 (-c)。函数skn-1应变 (lax188)的增益使 SKN-1 组成性激活 E237K 氨基酸替代。L4-stage 蠕虫暴露在0、15和 30 J/米2紫外光之后, 在 NR 染色和成像前非种子选手 NGM 板上有12小时的恢复期。显示的图像是表型的代表。脂质量化是按第1.7 节所述进行的。火是一个 ImageJ 的查找表 (查找), 用于创建一个显示图像之间荧光强度差异的热图像。合并显示 FITC/GFP 和 DIC 图像相结合。图像蒙太奇的底部显示了每个图像的荧光强度量化。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57352/57352fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57352/57352fig2.jpg"> 图 2: 油脂的染色由油红色 O.油红色 O 染色激活的skn-1突变体 (lax188) 显示日本自卫队表型 (A D) 的存在或缺失。& D 显示具有中间日本自卫队表型的动物。B & C 显示在破产检测对面。当 B 为非日本自卫队时, C 显示大量的躯体脂肪耗竭伴随增加生殖脂肪积累, 这定义了日本自卫队表型。这些蠕虫染色后, 144 hafter L1-synchronized 动物被放置在 OP50 细菌种子 NGM 培养基上。显示的图像是具有或没有日本自卫队表型的动物的代表。嵌入卡通被修改从林恩,等等。6 , 表示躯体脂肪耗尽的缺席 (B) 或存在 (C)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57352/57352fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

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

20.0K 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

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

科研

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

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 ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法:同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The carotid bodies (CB), a key organ of peripheral hypoxic sensitivity, express the long functional isoform of leptin receptor (LepRb) but the role of leptin signaling in control of breathing has not been fully elucidated. We examined the hypoxic ventilatory response (HVR) (1) in C57BL/6J mice before and after leptin infusion at baseline and after CB denervation; (2) in LepRb-deficient obese db/db mice at baseline and after LepRb overexpression in CBs. In C57BL/6J mice, leptin increased HVR and effects of leptin on HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB augmented the HVR. Therefore, we conclude that leptin acts in CB to augment responses to hypoxia.

Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed &#39;loss of function&#39; experiments measuring the effect of leptin on HVR after CB denervation and &#39;gain of function&#39; experiments measuring HVR after overexpression of the leptin receptor in CB.

检查胡萝卜体瘦素信号的实验方法及其对呼吸控制的影响

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The ...

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 ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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 ...