我们要感谢唐纳德准备和凯文·摩西教授JPK原成虫盘解剖过程。我们也感谢邦妮Weasner的生殖器盘图1B和眼盘图2A，布兰登Weasner为图3中，布鲁明顿果蝇库存中心飞污渍和发展研究杂交瘤细胞银行抗体。 CMS已经由卫生研究院（NIH）的GCMS培训格兰特国家研究院​​（T32-GM007757），弗兰克·W·普特南研究奖学金，以及罗伯特·布里格斯研究奖学金助学金的支持。 JPK支持由国家眼科研究所资助（R01 EY014863）

1，准备幼虫<ol><li>填写35mm的培养皿夹层缓冲。 </li><li>幼虫放置在培养皿中，让它们游来游去几分钟（自清洁步骤）。 </li><li>幼虫转移到夹层缓冲池的有机硅系夹层板。这个池应该是在该板的一个边缘。夹层板包括已注入硬化内的玻璃培养皿中的硅解决方案。 </li><li>使用巴斯德，移液管，将夹层缓冲池更大的夹层板的中间。 </li><li>使用＃5镊子，从小型游泳池传输单个清洗幼虫解剖缓冲区较大的游泳池。 </li></ol>幼虫2，粗剖析<ol><li>而幼虫仍然是夹层缓冲的大型游泳池内扣用钳子幼虫。一对钳子的，应使用抢口钩，而另一对镊子是用来装在ANIM人还在（抢轻轻的幼虫在人体三分之一的长度）。 </li><li>保持稳定的一对含角质层口附近的钩子，同时迅速与第二对钳子的提拉身体的其余部分离开钳子。 </li><li>当幼虫开始撕开，你会感到轻微的释放紧张情绪。从钳子松开幼虫，并允许幼虫洒出的“胆”。这允许对成虫盘留在其正常形态，防止它们变形。 </li><li>有一对钳子抓住钩嘴再次举行的地方幼虫的前端。使用其他钳子取出幼虫包括内胆量的低三分之二。注意：含有口钩，眼，触角光盘，大脑半球，腹侧神经节，唾液腺，某些腿光盘的复合物，以及上覆的角质层会维持不变。 </li><li>一对镊子轻轻取出上层的角质层，唾液腺，腿盘和其他组织。注意：只有组织应保留的口钩，眼，触角光盘，大脑半球，和腹侧神经节（ 图3）。 </li><li>额外的幼虫15-20分钟重复步骤2.1-2.5。注意：留在夹层的缓冲液的时间较长成虫盘趋向于降低，并最终将出现不理想的标本被照相。因此，最多20分钟的所有解剖组织中应该转移到PLP固定液后（见下文）。 </li></ol> 3，固定和组织染色的抗体<ol><li>使用的是P-200的Pipetman，解剖组织转移到含冷多聚甲醛赖氨酸高碘（PLP）固定液的手表玻璃。转夹层缓冲限位量50μL，以减少稀释PLP的。一定要切断的前端用刀片，使针尖开口大到足以容纳解剖组织。用钳子或钨针确保在解剖组织是为了正确固定到发生完全淹没。孵育在寒冷的PLP固定液解剖组织45分钟。该温育可以在RT发生不搅拌。 </li><li>使用的是P-200的Pipetman，使用另一种切割黄色针尖45分钟转移解剖组织，以洗涤缓冲液（RT）。转移PLP的极限量50μL，以减少稀释的洗涤缓冲液中。注：所有的解剖组织应完全淹没。 </li><li>转让20-30套解剖组织的1.5 ml离心管中。注意：如果较大的眼触角光盘络合物号在管内结合时，有可能使组织在管的底部将不会正确地暴露于抗体。为了达到最佳效果不会超过20-30眼触角盘配合应该是一个单一的管内出现。的解剖组织会沉淀到离心管的底部。因此，在后续步骤ü证A的Pipetman拆卸和更换洗涤缓冲液，封闭液和抗体。 </li><li>除去洗涤缓冲液和替换用100μl封闭溶液：在洗涤缓冲液中的10％正常山羊血清。在室温下孵育与在桌面上旋转10分钟，轻柔旋转。 </li><li>除去阻断溶液并更换用100μl一级抗体的已在10％正常山羊血清被适当地稀释。在室温下孵育以16小时平缓旋转。 </li><li>除去第一抗体，并替换为750微升的洗涤缓冲液。放置管上的章动器，并允许作俯仰运动在室温10分钟。注意：一次抗体可以被保存（储存在4°C）和后重复使用。重复使用的抗体多次可以帮助减少非特异性结合。 </li><li>允许磁头沉降到管的底部，然后除去洗涤缓冲液中，并加入100微升已经在正常血清中的10％被适当稀释的二抗的。在室温下孵育与温柔的转动2̵1; 4小时。 </li><li>从组织去除二级抗体溶液和替换用500μl的洗涤缓冲液。允许组织沉降到管底。 </li><li>使用的是P-200和切割黄色尖，全解剖组织转移到洗涤缓冲液已被放置在解剖菜池。而在进行细夹层的下一个步骤中，组织将孵育在洗涤缓冲液中。这有助于去除过量的二抗。 </li></ol> 4，精细解剖眼睛，触角盘配合，并安装到幻灯片<ol><li>用一对镊子的口钩朝下的复杂的腹侧扣着角质层。与第二对钳子的，通过关闭第2钳子在脑和眼盘之间的空间（ 图3，红色箭头），迅速 ​​地从口中钩拉大脑远离除去两个脑叶。 </li><li>同时继续稳守口钩，夹断组织尽可能接近到眼触角盘的触角部和口钩（ 图3，蓝色箭头）之间的连接。注：眼触角盘配合应无组织（ 图1A）。继续进行，直到所需的所有期望的组织可以放置在载玻片上已被切开。此协议可适应隔离翼，haltere，腿和生殖器成虫盘（ 图1B-E）。 </li><li>在解剖盘加两小块纸巾（盖玻片的大小）约3英寸分开。将载玻片上的菜 - 两片纸巾应该是幻灯片的目的下。这将防止在滑动粘附到解剖盘的硅氧烷基。 </li><li>利用P-20和未切割尖端，添加9微升抗漂白剂的玻璃显微镜载片上的中间。这防止了组织的漂白和荧光观察时光。 </li><li>使用相同的完整无缺的一角，收集所有的眼睛触角光盘，并将其添加到抗漂白剂的下降。最小化的所承载的洗涤缓冲液的量。试图限制的洗涤缓冲液的量，以10微升或更小。 </li><li>用一对镊子的分离和抗褪色试剂的滴内摊开眼触角光盘。允许光盘，以在抗褪色试剂孵育数分钟。注：光盘将变为清晰的组织吸收的抗漂白剂。 </li><li>用细的画笔轻轻放下盖玻片上的样品。这防止了气泡的形成。 </li><li>商店滑动于-20℃，直到准备查看使用光或荧光显微镜眼触角或其他成虫盘。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atonal,+rough+and+the+resolution+of+proneural+clusters+in+the+developing+Drosophila+retina.">Atonal, rough and the resolution of proneural clusters in the developing Drosophila retina.</a> Development. 122 (12), 4139-4147 (1996).</li><li>Kumar, J. P., Moses, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+Receptor+and+Notch+signaling+act+upstream+of+Eyeless/Pax6+to+control+eye+specification.">EGF Receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification.</a> Cell. 104, 687-697 (2001).</li></ol>

The authors declare that they have no competing financial interests.

虽然这个过程在很大程度上集中在分离和后续治疗的眼触角光盘，它是适合于被用于分离和分析的翼，haltere，腿和生殖器光盘（ 图4）。该协议用于分离这些光盘（相对于眼触角光盘）的唯一必需的修改是粗解剖（该协议的第2部分）的方法。第一胸椎腿（T1）对被发现在幼虫的前，可以通过以下的协议眼触角盘隔离来回收。然而，第二胸椎腿（T2）的光盘安装到角质层。以隔离这些光...

一个多世纪的果蝇， 果蝇 ，一直是首屈一指的系统来研究发展，行为和生理。发展中的苍蝇可分为两大阶段：胚胎和胚后得多，后者正在发生的范围内的单层上皮称为成虫盘1-3。 成虫盘的图纸首次出版于1864年八月魏斯曼作为他的专着广泛的昆虫开发1份 。这些光盘胚胎发育过程中开始了他们的发展，在幼虫阶段进行构图，生存下来的早期蛹阶段的大规模histolysis，最终导致成年结构被发现在成人中飞往1-14比例很高。在幼虫发育每张光盘公司对于命运，形状和大小的几个关键决策。在第一和第二龄幼虫，光盘的任务是采取主的命运，establis兴隔室的边界，采取了正确的形状和单元格生成15-16的必要数量。在第三龄幼虫和早期预蛹期，成虫盘继续分化和图案化为细胞通过它们的终端命运16。 在果蝇发育生物学的早期历史，成虫盘是在正常发展的情况下，并在有限的情况下，其中的损失或获得功能的突变体是可行的研究几乎完全。利用X射线来诱导允许致死突变有丝分裂重组到在幼虫和成体组织内的细胞克隆进行分析。此方法已被引入转基因方法提高分析损耗和增益的函数在两个幼体和成体组织中的突变。抗体，转录记者和蛋白质陷阱用于说明野生型和突变型组织中的分子景观的数目也是常量antly增长。使用这些分子标记分析损耗和增益的函数的突变体细胞克隆已经变得日益可行获得的如何从他们的野生型表亲发育过程中的突变细胞偏离实时了解。要正确利用这些工具和试剂，关键是要具有可观察，拍照和分析成虫盘高品质的准备。该原稿的目的是提供一种用于眼触角圆盘复合物（ 图1A）的分离和制备的优化协议。它也可以被成功地用于分离各种各样的另外的光盘包括那些引起的翅膀，halteres，T1-T3的腿和生殖器（ 图1B-E）。这个过程，有稍作修改，已用于隔离成虫盘果蝇了近80年。 如上所述，由于大多数的基因中亩表达ltiple发展，在许多组织中的阶段，它往往是不可能的学习效果无效突变对整个眼睛的动物的三龄幼虫期前去世好。四种方法取得了比较发达的组织学习，如视网膜显著更容易处理。第一个是一个否则野生型组织17-19内产生突变体细胞克隆的翻转酶（FLP）/翻转酶的重组目标（FRT）的方法。在此实例中，突变体组织是由缺少可视标记的识别，例如绿色荧光蛋白（GFP），并且可以比周围的野生型组织中的GFP存在（ 图2D）。第二是其中的转基因表达在细胞20的人口的“FLP出”方法。在此实例中的细胞克隆是通过GFP的存在鉴定和比周围的野生型组织的缺乏GFP报告（ 图2E）。三是具有可抑制细胞标记（MARCM）技术，它结合了FLP / FRT突变克隆和FLP出表达系统21元素的马赛克分析。用这种方法的转基因可在细胞，同时突变为单个基因座的群体来表达。像FLP出克隆，MARCM克隆通过GFP的存在鉴定和比周围的野生型组织的缺乏GFP标记（ 图2F）。最后，该基因和RNAi构建体可以特异性启动子-GAL4构建体的控制下，成虫组织中表达。这四种方法都增加学习成虫盘的兴趣，因为突变或过度表达克隆或图案可以直接比较，以相邻的野生型组织。在此过程中描述的方法已经被开发，使研究人员谁研究成体组织中的后胚胎发育的果蝇 ，特别是那些从眼触角光盘衍生，将能够获得高品质的组织进行分析。虽然个别研究人员已经作出轻微的修改，这个过程（其中我们在这里描述）的核心基本上保持不变。由于获得高品质的组织是在成虫盘，我们希望这份书面协议，并伴随视频将作为宝贵的教学资源的研究是至关重要的。

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Name of Material/Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:165px;">Catalog Number</td>
			<td style="width:360px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Used to create base for dissection plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pryex Glass Petri Dish 150x20mm</td>
			<td>Dow Corning</td>
			<td>3160-152CO</td>
			<td>Use the cover for dissection plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5 Dissecting Forceps</td>
			<td>Ted Pella</td>
			<td>525</td>
			<td>Forceps must be kept very sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9 well watch glass</td>
			<td>Vairous Vendors</td>
			<td>N/A</td>
			<td>Used for fixation of imaginal disc complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml Erlenmeyer Flask</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Stir Bar</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Small enough to fit into Erlenmeyer Flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml Conical Tubes</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5ml Microfuge Tubes</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Clear or Dark depending upon application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge Rack</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop Rotator</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>100ul volume should not splatter at low setting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Macron Chemicals</td>
			<td>2-26555-1</td>
			<td>Serves as fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate Monobasic</td>
			<td>Sigma Chemical Co</td>
			<td>S-3139</td>
			<td>Used to make dissection and wash buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate Dibasic</td>
			<td>Sigma Chemical Co</td>
			<td>71636</td>
			<td>Used to make dissection and wash buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysine</td>
			<td>Acros Organics</td>
			<td>125221000</td>
			<td>Used in the fixative solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Periodate</td>
			<td>Sigma Chemical Co</td>
			<td>S-1878</td>
			<td>Used in the fixative solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>MTX1568-1</td>
			<td>Used to perforate imaginal discs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>EM Science</td>
			<td>SX0593-3</td>
			<td>Used to dissolve paraformaldehyde</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Normal Goat Serum`</td>
			<td>Jackson Laboratories</td>
			<td>005-000-121</td>
			<td>Serves as a blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Antibodies</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Dilute in 10% goat serum as directed by manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seondary Antibodies</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Dilute in 10% goat serum as directed by manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield</td>
			<td>Molecular Probes</td>
			<td>H-1000</td>
			<td>Prevents bleaching of samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>Fischer Scientific</td>
			<td>48312-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Cover Slips 18x18mm</td>
			<td>Fischer Scientific</td>
			<td>12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe Tissue</td>
			<td>Various Vendors</td>
			<td>NA</td>
			<td>Prevents Glass slides from adhering to silicone base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Panit Brush 000</td>
			<td>Various Vendors</td>
			<td>NA</td>
			<td>Use to gently lower coverslip on to samples</td>
		</tr>
	</tbody>
</table>

dissection and immunostaining of imaginal discs from drosophila melanogaster

后期胚胎发育中的果蝇， 果蝇的显著部分，需要在一组囊样结构，称为成虫盘的地方。这些光盘引起成人结构的成年果蝇中发现的高百分比。在这里，我们描述了优化，回收这些光盘并准备分析的抗体，记者转录和蛋白质陷阱的协议。此过程是最适合用于薄的组织像成虫盘，但可以很容易地修改为用较厚的组织使用，如幼虫脑和成年卵巢。书面协议和相应的视频将通过三龄幼虫，组织固定和治疗成虫盘与抗体的夹层引导读者/观众。该协议可以用于解剖成虫盘从年轻的第一和第二龄幼虫为好。这个协议的优点是，它是比较短的，它有可连接优化的高品质保存的解剖组织。另一个优点是，所采用的固定方法可以很好地承认果蝇蛋白质的抗体的数量相当多。根据我们的经验，有极少数不以这个程序很好地敏感的抗体。在这种情况下，补救似乎是使用替代固定的鸡尾酒，同时继续遵循我们提出的解剖步骤和抗体孵育的准则。

这是上面描述可靠的方法产生高品质的材料进行分析原位探测，记者转录，蛋白质陷阱和抗体。在图1中，我们显示了例行回收用这种方法眼睛天线，生殖器，翼，haltere和腿部光盘。这些光盘已经处理了鬼笔环肽标记的荧光基团，其结合到F-肌动蛋白，从而勾勒每一个细胞。如果组织已被妥善固定，然后在眼盘的形态发生沟，同心组织的边缘褶皱的生殖器官，触角和腿盘，以及?...

Watch this Scientific Journal Video about Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster at JoVE.com

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

dissection-immunostaining-imaginal-discs-from-drosophila

Research

JoVE Journal

Biology

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

26.3K 次观看.  Indiana University. The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

视频: Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...