我们要感谢工对这个项目的财政支持, 对玛丽安娜 Di Doménico 为她的技术援助与共焦显微镜;Báez 为他的手稿修正;为他献身于创造视频的科雷亚;Rosano 为视频和布卢明顿果蝇股票中心提供了本研究中使用的股票的声音。Apa1 单克隆抗 Ptc 开发的伊莎贝尔格雷罗是从发展研究杂交瘤银行 (DSHB) 的主持下, 研究院和维护由爱荷华大学, 生物科学系, 爱荷华市 IA 52242。

1天
1. 收集和培养蛹。
<ol>
	<li>在蛹形成 (APF) 或蛹后, 使用湿的画笔从培养的小瓶中取出0小时, 并将它们放入新的瓶子中, 以几乎完成开发期 (见2.1 和 2.2)。 
	注意:应该使用标准的培养规程来维护健康的苍蝇种群。在产卵三或四天后, 成人可以从小瓶中取出, 以使个体得到最佳的发育。他们保持在恒定的温度, 直到第三个幼虫开始蛹。幼虫/蛹的转变是以蛹的形成为标志的, 被认为是 0 h APF。蛹是一个不动的白色幼虫, 有一个长方形, 圆形, 突出前气孔。</li>
</ol>
2天
2. 转移、解剖和固定。
<ol>
	<li>将蛹 (2 小时前的开发时间完成) 与一支画笔, 到显微镜幻灯片与双面胶带坚持它。使蛹形成一排背侧和头端朝向同一方向。</li>
	<li>将显微镜幻灯片返回到恒定温度, 并允许蛹完成适当的发育时间 (即24-30 h APF)。 
	注意:与双面胶带的显微镜滑动将是一个暂时的解剖平台, 以消除蛹的情况下 (见下文)。显微镜滑动 (瓶外) 的时间推移有助于干燥蛹的情况下, 使其容易破碎的钳子。</li>
	<li>用镊子将盖从蛹箱中取出。</li>
	<li>用钳子 (如视频中所示) 打破这一情况, 沿着蛹的一侧到它的尾端线。有了适当的光照, 就可以检测蛹的内部形状, 使其在蛹翼的铰链上方有一个明显的空间。这个空间作为一个指南, 以执行开放, 而不损害蛹。</li>
	<li>删除部分的情况下, 拾起他们在打开的边界, 并携带他们到对面的双面胶带将举行他们。在解剖的最后, 蛹几乎是 "赤裸", 只有一小部分的情况下将覆盖后端。 注: 在上尾端留下少量的情况是为了确保从机箱上平滑地释放到带有双面胶带的滑块上。这一行动背后的原因是, 如果你删除太多的情况下, 你很大的风险损害蛹。另一方面, 如果你删除太少, 蛹将不容易来摆脱的情况。</li>
	<li>用镊子把箱子的其余部分往下推。这一运动将提高蛹的前端, 使头部和胸部保持在一个优先地位, 以坚持和转移在随后的步骤。</li>
	<li>用双面胶带将新的幻灯片翻转到蛹或蛹排。我们建议采取这种方法, 观察运动的帮助下, 显微镜防止蛹的破坏。</li>
	<li>稍微按压一下, 使蛹粘在胶带上, 然后轻轻地滑动, 以便从其余的病例中取出蛹。倒置显微镜幻灯片: 蛹将被卡在背侧的水平, 他们的头部/胸部区域。</li>
	<li>覆盖所有的裸蛹与4% 甲醛和等待10分钟。根据我们的经验, 这种固定的时间的推移有助于改变角质层的一致性, 使其牢固, 不粘, 易于处理。 
	注意:为了执行核糖核酸 (RNA) 提取, 替代甲醛的磷酸盐缓冲盐水 (PBS) 制成的核糖核酸自由水和删除的蛹翅膀作出削减 (借助剪刀) 的铰链点附近的每个附属物。用镊子将蛹翼转移到离心管上, 用1毫升的硫氰酸胍和苯酚试剂。收集了总共50-80 个机翼后, 将离心管存储在-80 ° c, 直到执行 RNA 提取10,11。</li>
	<li>在机翼的铰链区域或靠近它的地方做一个小切口。使定影液到达翼上皮。在机翼上使用吸管 (p200) 冲洗定影液。当冲洗取代被污染的执着与新。冲洗后, 将机翼固定在定影液中5分钟。 
	注意:虽然这种机动有助于清除大部分的污染组织, 一些血和脂肪滴可能仍然被困在背部和腹侧上皮层之间的机翼 (见图 1A)。</li>
	<li>撕裂与钳子从角质层边界 (如在视频中所示) 扩大孔, 并使释放的翼上皮从角质层囊。一旦机翼组织被释放, 留在甲醛溶液中完成30分钟的固定。</li>
	<li>将固定翼转移到一个满是 PBST (海卫 X-100 0.05%) 的4溢的塑料碟中。在4° c 过夜。</li>
</ol>
3天
3. 主要抗体的培养。
<ol>
	<li>Permeabilize 的组织孵化蛹翼在 PBST (海卫 X-100 0.2%) 为15分钟使用摇摆平台, 以促进温和的液体介质运动。 
	注意:在安装这些示例之前, 应使用摇摆平台执行这些步骤。</li>
	<li>使用 PBSTA (BSA 3%, 海卫 X-100 0.1%) 为1小时阻止样品。</li>
	<li>使用相同的缓冲液来稀释主抗体 (小鼠抗 Ptc, 1:100), 在4° c 的夜间孵育蛹翅。</li>
</ol>
4天
4. 二级抗体培养。
<ol>
	<li>洗涤样品 4 x 10 min 每个与 PBST (海卫 X-100 0.05%)</li>
	<li>孵育与二次抗体 (例如, anti-mouse 共轭到荧光) 稀释到适当的集中在 PBSTA (BSA 3%, 海卫 X-100 0.1%) 在室温下在黑暗中, 为 2 h。 
	注意:如果合适, 使用 4 ", 6-diamidino-2-吲 (DAPI) 和/或笔 counterstain 的组织。将这些探针与二次抗体结合, 考虑到荧光的发射波长, 以防止信号重叠。</li>
	<li>洗涤样品 4 x 10 min 每个与 PBST (海卫 X-100 0.05%)</li>
</ol>
5. 安装
<ol>
	<li>在显微镜上放置4点透明指甲油, 准备一个样品幻灯片, 就好像它们是正方形的顶点一样。</li></ol>他们将是片将休息, 帮助避免粉碎组织的点。
	<li>放置30µL 的安装介质 (甘油/三-HCl 80%) 在广场中心的点, 用指甲油, 而不触及任何角落 (点)。取一个带有黄色裁剪尖端的吸管, 装上一些安装介质, 然后用几个蛹的翅膀画出。</li>
	<li>将蛹的机翼转移到显微镜滑轨上的安装介质的点上。在尖端的帮助下, 传播安装媒体和下沉蛹的翅膀, 以防止他们漂浮。当盖上玻璃被放置时, 如果机翼保持平坦, 组织的形态将保持不变。在放置盖子玻璃之前, 除去气泡。</li>
	<li>将片放在样品上, 试图防止气泡的产生。这一运动可以借助镊子来完成。</li>

<ol>
	<li>Serrano, N., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+morphogenesis:+connections+between+patterning+and+growth.">Limb morphogenesis: connections between patterning and growth.</a> Curr Biol. 7 (3), R186-R195 (1997).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+appendage+development+in+Drosophila.">Compartments and appendage development in Drosophila.</a> Bioessays. 17 (4), 299-309 (1995).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.">Wing disc development in the fly: the early stages.</a> Curr Opin Genet Dev. 11 (4), 470-475 (2001).</li><li>Beira, J. V., Paro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+legacy+of+Drosophila+imaginal+discs.">The legacy of Drosophila imaginal discs.</a> Chromosoma. 125 (4), 573-592 (2016).</li><li>De Celis, J. F., Diaz-Benjumea, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+basis+for+vein+pattern+variations+in+insect+wings.">Developmental basis for vein pattern variations in insect wings.</a> Int J Dev Biol. 47 (7-8), 653-663 (2003).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+vein+patterning+in+Drosophila+and+the+analysis+of+intercellular+signaling.">Wing vein patterning in Drosophila and the analysis of intercellular signaling.</a> Annu Rev Cell Dev Biol. 23, 293-319 (2007).</li><li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drawing+lines+in+the+Drosophila+wing:+initiation+of+wing+vein+development.">Drawing lines in the Drosophila wing: initiation of wing vein development.</a> Curr Opin Genet Dev. 10 (4), 393-398 (2000).</li><li>De Celis, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+wing:+The+development+of+the+veins.">Pattern formation in the Drosophila wing: The development of the veins.</a> Bioessays. 25 (5), 443-451 (2003).</li><li>Classen, A. K., Aigouy, B., Giangrande, A., Eaton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Drosophila+pupal+wing+morphogenesis.">Imaging Drosophila pupal wing morphogenesis.</a> Methods Mol Biol. 420, 265-275 (2008).</li><li>Bolatto, C., 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=Spatial+and+temporal+distribution+of+Patched-related+protein+in+the+Drosophila+embryo.">Spatial and temporal distribution of Patched-related protein in the Drosophila embryo.</a> Gene Expr Patterns. 19 (1-2), 120-128 (2015).</li><li>Zuniga, A., 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=Genes+encoding+novel+secreted+and+transmembrane+proteins+are+temporally+and+spatially+regulated+during+Drosophila+melanogaster+embryogenesis.">Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis.</a> BMC Biol. 7, 61 (2009).</li></ol>

作者没有什么可透露的。

该视频中所描述的解剖方法可用于为许多类型的技术 (如,免疫荧光法, cDNA 合成, PCR 检测) 制备高质量的果蝇蛹翅的样品. 体外翼发育)。根据这种方法, 科学家们使用了 24-30 h 型 APF。然而, 在解剖年轻或年长的蛹的关键步骤中, 不应有任何障碍。可能必须作出改变, 以适应较小的阶段蛹。
该协议可以进行解剖一个蛹或几个蛹在同一时间。这有助于加快过程, 使它更容易到达大量的翅膀解剖。在这种情况下, 有选择删除一行4-5 蛹在同一时间。您可以将蛹一起移除, 或者如果不可能, 继续逐个移到同一张幻灯片上 (参见视频的第三部分: "多蛹解剖")。为了去除裸露的蛹从打开的情况下, 重要的是, 后端的尖端是足够小。可以完全删除该案件;虽然它会使转移速度更快, 整个案件的清除是具有挑战性的, 有更大的风险损害蛹。
经验表明, 一个重要的机动考虑是离开 (最后2小时完成规定的发展时间) 的蛹附着在显微镜幻灯片孵化器内, 但瓶外。这种时间的推移有助于干燥的情况下, 使其更容易打破。在解剖过程中, 免疫组织化学的另一个关键步骤是剥离覆盖翼上皮的角质层, 从而使抗体进入整个机翼表面。此步骤应在固定几分钟后完成 (在铰链点切口后至少5分钟), 因为定影液稳定了组织, 改变了角质层的一致性, 使其更容易剥离。
在执行所有免疫组织化学步骤时, 确保机翼在恒定运动 (摇摆平台)。该运动有助于改善在洗涤和孵化的解决方案的交流和渗透。然而, 重要的是要执行它们轻轻地避免在这个脆弱的结构的形态学不受欢迎的扭曲 (即,它是足够的振动平台轻微移动的液体介质包含样本)。
这一程序提供了一种替代和补充的方法, 已经公布的方法, 蛹翼解剖。

果蝇机翼是从机翼形象盘中开发的。在胚胎发育过程中, 这些翼盘的原始性被放在一边, 作为20-30 形象细胞的小基团, 从胚胎上皮 invaginate。当幼虫生长到第三个龄期时, 有丝分裂发生在形象细胞在特定的特征时间提高其数量 (约50000细胞), 形成翼盘1,2,3, 4。当细胞增殖时, 它们会形成管状上皮, 形成 self-folding 的致密螺旋。在蛹期间, 圆盘上皮望远镜形成二层翼和纵脉开始作为他们之间的空白, 在翼发展期间发生两次5,6。
静脉的排列总是有相同的图案;能够很容易地识别血管形成中的每一个变化, 使得突变非常明显 (例如缺失或额外的静脉, 静脉位置的变化,等)。有趣的是, 表型变化通常是已知或新奇的信号传导通路的突变的证据, 它们与机翼的发育有关。在确定位置和维持静脉和交错区域中扮演角色的一个途径是刺猬 (Hh) 路径6,7,8。
考虑到蛹机翼作为一个模型系统的重要性, 获得高质量的样品是很重要的。过去, 科学家们已经发布了解剖果蝇翅膀的协议, 但并没有给出合适的指南来获得可行的样本。没有研究员的教导你不可能清楚地形象化过程。在这些交替的解剖方法中, 蛹被一分为二, 将机翼与周围的组织隔开, 并反复洗涤以除去碎片。这种方法声称离开机翼的污染, 但由于以往的经验的过程是较慢的, 有更大的机会破坏脆弱的机翼结构9。
这里描述的程序是根据需求找到一种快速和有效的方法获得蛹翼样本, 适合检测特定的蛋白质或抄本使用免疫协议或聚合酶链反应 (PCR) 化验。为了说明这一点, 在蛹翼样本中检测到了已修补的 (Ptc 或 Hh 通路的典范受体) 的表达和定位, 提供了包含相关细节的协议, 以成功地完成 程序.

<table border="0" cellpadding="0" cellspacing="0" width="809">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Stereomicroscope with cool light illumination</td>
			<td style="width:175px;">Nikon&#160;</td>
			<td style="width:119px;">SMZ1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rocking platform</td>
			<td>Biometra (WT 16)</td>
			<td>042-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides</td>
			<td>StarFrost</td>
			<td>8037/1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover glasses (24 x 32 mm)</td>
			<td>Deltalab</td>
			<td>D102432</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Lawton</td>
			<td>63-1400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multi-well plastic dish</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">p100 or p200 pipette</td>
			<td>Finnpipette (Labsystems)</td>
			<td>9400130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1X PBS (Dulbecco)</td>
			<td>Gibco</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Bio Basic INC.</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Mallinckrodt</td>
			<td>5092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Amresco</td>
			<td>497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TRIzol Reagent</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DEPC treated water</td>
			<td>MO.BIO</td>
			<td>17011-200</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:349px;">Alexa Fluor 546 goat anti-mouse IgG (H+L), 2 mg/mL 
			(working dilution 1/800)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11030</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="153">
			<td height="153" style="height:153px;width:349px;">Monoclonal anti-Ptc (Apa-1) 54 &#956;g/mL 
			(working dilution 1/100)</td>
			<td style="width:175px;">Developmental Studies Hybridoma Bank (DSHB)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">By Isabel Gerrero from DSHB under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:349px;">DAPI, 1 mg/mL 
			(working dilution 1/5000)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">62248</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:349px;">Phalloidin conjugated to Alexa 488, 300 U/1.5 mL methanol 
			(1/600 working dilution)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:349px;">Canton S (fly stock)</td>
			<td style="width:175px;">Bloomington Drosophila Stock Center&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a rapid and efficient method to dissect pupal wings of drosophila suitable for immunodetections or pcr assays

在果蝇的机翼发育是研究组织层形态发生的理想模型。这些附属物从胚胎发育过程中形成的一组叫做翼形象的细胞发展而来。在幼体阶段, 形象椎间盘生长, 增加细胞数量, 形成 monolayered 上皮结构。在蛹的情况下, 形象盘芽和折叠成双层沿一条线, 成为未来的翼缘。在这一过程中, 纵 primodia 脉在翼的准背和腹面上起源于静脉细胞。在蛹阶段, 每个表面的静脉细胞的条纹, 以产生致密管;与此同时, cross-veins 开始形成。
在适当的分子标记的帮助下, 它是可能的确定主要元素构成翼在它的发展期间。由于这个原因, 能够准确地检测到该结构中的转录或蛋白质, 对于研究它们的丰度和定位与机翼的发展过程是至关重要的。
这里介绍的过程集中在操纵蛹翼, 提供详细的说明如何解剖机翼在蛹阶段。蛹组织的解剖比它们在第三龄幼虫中的表现更难。这就是为什么这种方法的发展, 以获得快速和高效的高质量样品。有关如何免疫和安装这些机翼样品, 让蛋白质或细胞成分可视化的细节, 在协议中提供。由于缺乏专业知识, 可以在短时间内收集8-10 高品质的蛹机翼。

该协议提供了一个简单的方法获得蛹翼样本, 保留了熟悉的机翼形态。从这些准备工作中, 可以获得一堆可能被投射为2维或3维的图像, 以研究在机翼分化过程中蛋白质的分布和/或丰富度。类似的协议 (见2.9 的说明) 可以用来收集大蛹翼样本 (涉及50-80 蛹翼) 合成互补脱氧核糖核酸 (cDNA), 用于 PCR 反应的模板。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55854/55854fig1.jpg"> 
图 1: 刺猬路径基因在果蝇蛹翅膀 
(A)补丁, 刺猬路径的典范受体, 在 24-30 h APF 中使用小鼠抗 Ptc 进行了可视化. 细胞核与 DAPI 1-4, 而肌动蛋白丝则用绿色荧光笔。肌动蛋白分布有助于定位翼脉及其与已修补表达式的关系。与此同时, 笔染色揭示了一些血和脂肪滴 (大圆圈) 的存在, 作为污染组织。20x 放大倍数, 前部向上。用类似的解剖协议生成的信使 RNA (mRNA) 翼样本, 利用 cDNA 综合获得了100µm (B)增到肌动蛋白(车道1、280 bp) 和ptc (lane 2, 236 bp) 的比例。兆瓦, 分子量。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55854/55854fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays at JoVE.com

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

在果蝇组织中准确检测转录和蛋白质的能力对于研究它们与发展过程有关的丰度和定位是至关重要的。这里是一个简单的过程解剖蛹翅膀的描述。这些翅膀可以作为样品在一些技术 (免疫组织化学, PCR 检测,等)。

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

a-rapid-efficient-method-to-dissect-pupal-wings-drosophila-suitable

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

8.0K 次观看.  Laboratorio de Biología del Desarrollo. 在果蝇组织中准确检测转录和蛋白质的能力对于研究它们与发展过程有关的丰度和定位是至关重要的。这里是一个简单的过程解剖蛹翅膀的描述。这些翅膀可以作为样品在一些技术 (免疫组织化学, PCR 检测,等)。

视频: A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...