我们感谢 AQUAGAMETE 1205 的成本行动。这项工作得到了 AGL201568330-C2-1-R 项目 (MINECO/菲德) 的财政支持。大卫 g. Valcarce 由 EDU1084/2012 和 Fondo 社会 Europeo 资助。作者承认安娜 Riaza 博士和 Stolt 海洋农场, Paulino 博士, 拉巴斯博士蒙特罗和何塞拉蒙 Guiérrez。我们还感谢宝拉. 费尔南德斯. Colado 为录像。

注: Fluorochrome 孵化和共焦分析将采取至少2-3 小时的控制和治疗样品。在这个时间计算中不包括数据处理。所需材料可在材料表中找到。本协议可应用于新鲜或冷冻保存的精子。Solea senegalensis是一种在冷水中产生的鱼类, 通常在寒冷的环境下工作 (4-7 摄氏度)。有关协议的一般视图, 请参见图 1 。
1. 实验前的准备工作
<ol><li>在分析级二甲基亚砜上制备1毫米线粒体染色液。</li>
	<li>在去离子水中制备1微克/毫升 4′-, 6-diamidino 2-苯基吲哚盐酸盐 (DAPI) 溶液。</li>
	<li>准备一个 200 mOsm/千克响铃解决方案 (116 毫米氯化钠, 2.9 毫米氯化钾, 1.8 毫米 CaCl2, 5 毫米 HEPES, pH 7.7)</li>
	<li>在分析级甲醇上制备4毫米-2′-、7′-dichlorodihydrofluorescein 双乙酸 (DCFH DA) 溶液。 注: 保持库存解决方案在整除数-20 摄氏度, 直到需要。</li>
	<li>将离心设置为4-7 摄氏度。</li>
</ol>2. Fluorochrome 孵化前的样品准备
注意: 当吹打和 resuspending 时, 对精子的温和处理是可取的。
<ol><li>如果使用保存的细胞, 准备一个水浴为解冻在40°c 为Solea senegalensis精子10。将秸秆样品浸入水浴中7秒。</li>
	<li>使用剪刀切割, 把样品空成一个离心管, 离心机在 1000 x g, 4-7 摄氏度为1分钟。</li>
	<li>用吹打从冷冻保存的样品中取出精浆或精浆冷冻保护剂, 丢弃任何可能干扰染色协议的成分。</li>
	<li>并用重悬细胞在 50-100 ul 4 °c 200 mOsm/公斤响铃解决方案。</li>
	<li>用 Neubauer 或类似的计数室在立体显微镜下测定精子浓度。</li>
	<li>将细胞悬浮液稀释至 1-2 x 106细胞/mL, 在4摄氏度的溶液中最终体积为0.5 毫升。</li>
</ol>3. Fluorochrome 孵化
注: 荧光必须在低光照条件下处理, 特别是在溶液中。
<ol><li>添加 3.125 ul DCFH-DA 股票溶液 (最后浓度在样品:25 微米) 到工作样品稀释。在黑暗中孵化4-7 摄氏度40分钟 (10。</li>
	<li>在30分钟以后, 增加 0.5 ul DAPI 存货解答 (最后的集中在样品: 2 微米) 和 0.5 ul 线粒体污点存货解答 (最后的集中在样品: 100 毫微米) 对样品。在黑暗中孵化两荧光10分钟。</li>
</ol>4. 共焦显微术的样品准备
<ol><li>潜伏期后, 离心机的细胞悬浮在 1000 x g, 4-7 摄氏度为1分钟。</li>
	<li>吹打小心地丢弃上清。</li>
	<li>添加 10-20 ul 的 200 mOsm/公斤的铃声解决方案。</li>
	<li>放置 5 ul 的浓缩细胞悬浮下降在幻灯片上。</li>
	<li>小心, 把封面幻灯片上的准备, 并密封它与波兰。一旦波兰人干了, 准备准备为共焦显微镜。</li>
</ol>5. 实验前共聚焦设置
注: 根据显微镜, DCFH 可能被 "烧伤"。先用非贵重样品建立最佳条件。
<ol><li>打开显微镜、激光、照相机和运行显微镜的电脑。</li>
	<li>设置励磁激光器, 考虑到每个 fluorochrome 的激发和发射最大度:<ol>
<li>对于 DAPI, 使用的激发最大值为 358 nm, 发射最大值为 465 nm。</li>
		<li>对于线粒体染色, 使用最多 644 nm 的激发和发射最大值为 662 nm。</li>
		<li>对于 DCFH, 使用的激发最大值为 504 nm, 发射最大值为 525 nm。</li>
	</ol>
</li>
	<li>开始使用25X 目标, 专注于精子。选择要集中的 DAPI 通道, 因为此 fluorochrome 报告的强度最高。一旦单元格集中, 请使用63X 或100X 目标进行彻底评估, 因为Solea senegalensis精子大小约为2微米。</li>
	<li>对于每个通道, 优化针孔, 增益和电压。同样地, 调整数字偏移量以减少背景。为所使用的 fluorochrome 优化所有参数后, 保存设置。对于单个Solea senegalensis精子细胞的常规实验, 我们使用了以下方法 (这些参数可能会在每个实验中发生变化): 针孔1和增益电压 750 v。</li>
</ol>6. 图像的采集
<ol><li>为图像获取选择所需的质量条件。定义采集设置, 如钻头深度, 图像格式, 光片厚度, 并选择单面照明。对于单个Solea senegalensis精子的常规实验, 我们使用了以下方法 (这些参数可能在每个实验中发生变化): 1024年像素的帧大小;位每像素 16;扫描速度为 2-4)。</li>
	<li>打开并居中扫描区域以开始并采取该字段的图像。</li>
	<li>使用裁剪工具, 选择感兴趣的区域并按 "实时"。</li>
	<li>在 "范围指示器" 工具的帮助下, 调整数字偏移量以减少背景。为每个通道执行此项, 并采取该图像。</li>
	<li>检查每个 fluorochrome 及其组合的各个通道的质量。</li>
</ol>7. 创建3D 图像视频
<ol><li>Z 堆栈捕获设置。<ol>
<li>在软件中选择 Z 堆栈选项。</li>
		<li>选择单个单元格、一组单元格或感兴趣的区域并将其聚焦。</li>
		<li>使用 "第一个切片" 和 "最后一个切片" 选项分隔 z 堆栈, 并在精细焦点的帮助下, 将 z 步骤设置为间隔为0.2 µm。选择 DAPI 频道集中, 因为这 fluorochrome 可能报告的最高强度, 你可以很容易地选择整个精子头。选择每个通道的最佳采集参数并运行实验。</li>
	</ol>
</li>
	<li>运行 Z 堆栈实验。拆分通道并观察单元格内每个信号的定位。</li>
	<li>Z 堆栈过程。<ol>
<li>卷渲染: 实验完成后, 选择 Z 堆栈处理软件的选项, 然后选择 "音量渲染" 选项。选择步骤数和旋转度数 (360°)。使用视频扩展名保存文件。</li>
		<li>立体声浮雕: 在 "选项" 过程窗口中选择 "立体声浮雕"。此工具允许使用分割通道创建3D 图像视频。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Amaral, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+and+chemical+compound+action+on+sperm+function.">Mitochondrial functionality and chemical compound action on sperm function.</a> Curr Med Chem. , (2016).</li><li>Morielli, T., O'Flaherty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+impairs+function+and+increases+redox+protein+modifications+in+human+spermatozoa.">Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.</a> Reproduction. 149 (1), 113-123 (2015).</li><li>G&#252;rler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. , (2016).</li><li>Zhu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+E+Analogue+Improves+Rabbit+Sperm+Quality+during+the+Process+of+Cryopreservation+through+Its+Antioxidative+Action.">Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.</a> PLoS One. 10 (12), e0145383 (2015).</li><li>Fang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ROS+production+through+mitochondria-targeted+antioxidant+and+mitochondrial+uncoupling+increases+post-thaw+sperm+viability+in+yellow+catfish.">Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.</a> Cryobiology. 69 (3), (2014).</li><li>Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A., Clark, 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=Cryopreservation-induced+human+sperm+DNA+damage+is+predominantly+mediated+by+oxidative+stress+rather+than+apoptosis.">Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.</a> Hum Reprod. 24 (9), 2061-2070 (2009).</li><li>Kim, S. H., Yu, D. H., Kim, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+phosphatidylserine+translocation,+intracellular+hydrogen+peroxide,+and+DNA+integrity+in+canine+sperm.">Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm.</a> Theriogenology. 73 (3), (2010).</li><li>Guthrie, H. D., Welch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reactive+oxygen+species+on+sperm+function.">Effects of reactive oxygen species on sperm function.</a> Theriogenology. 78 (8), 1700-1708 (2012).</li><li>Aitken, R. J., Jones, K. T., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+sperm+function--in+sickness+and+in+health.">Reactive oxygen species and sperm function--in sickness and in health.</a> J Androl. 33 (6), (2012).</li><li>Valcarce, D. G., Robles, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+captivity+and+cryopreservation+on+ROS+production+in+Solea+senegalensis+spermatozoa.">Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.</a> Reproduction. 152 (5), (2016).</li><li>Gibb, Z., Lambourne, S. R., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradoxical+relationship+between+stallion+fertility+and+oxidative+stress.">The paradoxical relationship between stallion fertility and oxidative stress.</a> Biol Reprod. 91 (3), (2014).</li><li>Cabrita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+enhancing+fish+sperm+quality+and+emerging+tools+for+sperm+analysis.">Factors enhancing fish sperm quality and emerging tools for sperm analysis.</a> Aquaculture. 432, 389-401 (2014).</li></ol>

作者没有什么可透露的。

众所周知, 线粒体是精子运动和功能的关键细胞器。这些细胞器同时直接参与 ROS 的生产。有趣的是, 适当的精子功能需要控制的活性氧含量为1。在哺乳动物中, 生育率和氧化应激之间的正相关关系已显示在11中, 但过度的水平影响精子质量12。一个关键的因素, 可能是决定性的积极或消极的影响, 不仅是 ros 水平, 而且 ros 细胞内定位。ros 的有害?...

活性氧种类的生产已与精子质量最近的1。虽然线粒体中的 ros 生产可以被认为是正常的生理过程, 但氧化应激是由过量的 ros 产生的, 是导致精子在不同水平上损伤的明显原因。在人类中, 氧化应激与男性不育、改变运动和接受精子2的能力有关;在哺乳动物中, 冷冻精子样品中 DNA 完整性的变化也与 H2O23的合成有关。
冷冻保存是水产养殖中的一种常用的基因银行技术。这种技术在具有生殖问题 (如Solea senegalensis) 的物种中尤为重要。这个市场上有价值的物种显示, 由于缺乏求爱, 在圈养的个体中繁殖功能失调。这一事实使精子的超低温保存必须有精子可用性的人工受精。然而, 冷冻保存可能是氧化应激的来源, 可能是有害的精子4 , 因为研究报告了有益的效果, 补充抗氧化剂。据报道, 通过线粒体靶向抗氧化剂的活性氧抑制有助于黄鲶鱼的精子超低温保存5。
因此, 精子样本中 ROS 的含量是很重要的, 特别是在保存6、7之后, 因为这些分子被认为是精子生存和生育能力8的一个缺点。此外, 研究细胞中 ROS 的分布对于推断潜在损伤的程度是至关重要的。例如, 线粒体中的低活性氧能被认为是正常的, 与精子功能相容, 但细胞核内的高活性氧含量可能是精子 DNA 损伤的指标。H2O2是最相关的 ROS 之一, 可以从线粒体中释放出来, 穿透细胞核, 因为它是一个小而无电荷的分子9。Dichlorofluorescein 双乙酸 (DCFH) 可以明确揭示胞内过氧化物发光的绿色荧光。本文介绍了用共聚焦显微镜检测Solea senegalensis精子中 H2O2细胞内定位的详细协议。

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA)&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6883</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8242;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MitoTrackerDeep Red&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>M22426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge (refrigerated)</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauerchamber</td>
			<td>Sigma-Aldrich</td>
			<td>BR717810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>10143562BEF</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa

氧化应激是降低精子质量的重要因素之一。开发有效的检测精子中活性氧 (ROS) 的协议在任何物种中都是非常重要的, 但是这些方法在硬骨鱼类中很少使用, 甚至更少。冷冻保存是水产养殖的一种有用的技术, 为不同的目的, 包括基因银行和保证精子的可用性全年。冷冻/解冻程序可能导致 ROS 的产生和损伤精子细胞。考虑到过量的 ROS 生产可能导致精子的潜在损害取决于它们的定位, 这里有一个详细的方法来检测 H2O2和评估其细胞内定位的共焦显微镜提供。为此, 采用3荧光 (-2′-、7′-Dichlorodihydrofluorescein 双乙酸 (DCFH DA)、活线粒体染色和4′-、6-Diamidino-2-苯基吲哚二盐酸盐 (DAPI)) 来评价H2 O 2 的协同定位与精子细胞核或线粒体在Solea senegalesis精子样本。

共焦显微术是硬骨鱼类精子细胞内 ROS 评价的理想方法。这项研究 (图 1) 中提出的三荧光 (DAPI、线粒体染色和 DCFH) 的组合提供了许多有用的信息, 可应用于基础研究, 并可应用于改进工业水产养殖植物, 如冷冻保存协议。可以进行不同类型的分析, 以关联细胞内 ROS 的存在和其他参数: 运动, 生存能力, 不同模式之间的良好和不良育种, 活化运动或季节性?...

Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

此协议描述了一个用于检测Solea senegalensis精子中的 H2O2本地化的详细方法, 使用敏感的 fluorochrome DCFH-DA 为 ROS、线粒体的活线粒体染色和细胞核的 DAPI可视化, 分别。该协议的设计是在2小时内进行, 无论是新鲜或解冻精子。

Solea Senegalensis精子中活性氧的细胞内定位评价

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

evaluation-intracellular-location-reactive-oxygen-species-solea

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Developmental Biology

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

6.2K 次观看.  University of León. 此协议描述了一个用于检测Solea senegalensis精子中的 H2O2本地化的详细方法, 使用敏感的 fluorochrome DCFH-DA 为 ROS、线粒体的活线粒体染色和细胞核的 DAPI可视化, 分别。该协议的设计是在2小时内进行, 无论是新鲜或解冻精子。

视频: Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

科研

发育生物学

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...