We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

1.解剖卵巢管的<ol><li>转约15 2-4日龄雌性果蝇和少数的男性用加入活性干酵母新鲜飞食品小瓶中。使用棉作为插件的小瓶，并添加几滴水到棉花，以保持湿度较高。 </li><li>在25℃的培养箱中14-16小时处小瓶最大化阶段8-10卵室的数目。 注：取决于温度和所需的阶段孵化时间会有所不同。 </li><li>制备夹层媒体，0.1M KPO 4和NP40的解决方案，根据需要，为第二天（见材料）。 </li><li>麻醉使用CO 2的苍蝇，和解剖显微镜下放置。吸管几滴夹层媒体到玻璃中的凹陷滑动每个空腔。 </li><li>持在每手钳在一个大约30°角，以该表顶部和东方1雌蝇的翅膀下。用你的非惯用手，拿起女性使用镊子，在一个抓她nterior腹部，靠近胸部，并淹没她进入了抑郁症的幻灯片（ 图1A插图）解剖媒体。 </li><li>与其它对镊子，把握外骨骼在腹部的腹后和刺穿。抢卵巢在后端，然后将它们拉出来腹部并放入新鲜培养基上的凹陷滑动的另一侧。 （ 图1A小图）。避免拉开肠道。丢弃的尸体。 </li><li>与其他雌性果蝇重复，直到所有的卵巢解剖。丢弃的男性。 </li><li>用一对镊子，保持在后端卵巢（靠近最大卵腔），并用另一只手用一对镊子夹住的最前蛋室（germarium）之一。 （ 图1A）。 </li><li>缓慢而稳步推进，拉动了卵巢管链出卵巢鞘。继续解剖出卵巢管单独直到所有所需的卵室为Removed。 </li><li>重复步骤1.7-1.8为所有解剖卵巢。 </li><li>完成后，用塑料移液管转移到卵巢管一管0.6毫升。 </li><li>让卵巢管沉降到试管底部，然后进行定影和染色步骤。 </li></ol>卵巢管2.免疫荧光（IF）染色<ol><li>小心地从卵巢管用吸管取出术媒体。一定不要删除任何卵巢管和蛋室。留出大约50微升卵巢管和解剖媒体。 </li><li>加入50微升16％多聚甲醛的150微升0.1M的KPO 4缓冲在管，并添加解决鸡蛋室，以解决这些问题。孵育而摇动10分钟。注意：多聚甲醛是有毒的。 </li><li>除去固定液和用0.5ml NP40洗涤缓冲液冲洗两次。 </li><li> 15每个在RT（分钟）洗两次。 </li><li>参考标准IF协议15后续步骤。 <ol><li>简单地说，固定后，加在适当的稀释度的一抗孵育O / N在4℃。在NP40几次洗涤，然后加入第二抗体（1：200稀释度）。 </li><li>在NP40洗几次，然后加入DAPI（1：1000）进行10分钟，然后重复洗涤。一旦中频协议完成后，在PBS中加入200微升50％甘油的蛋腔室和让坐在在RT搅拌2小时。 </li><li>除去溶液，用在PBS中的70％的甘油代替，并盖上箔。将样品在4ᵒC直到准备安装。同时，继续执行步骤3。 </li></ol></li></ol>甘油果冻幻灯片3.事前准备<ol><li>测量至少4甘油果冻用抹刀毫升，并填补了玻璃细胞培养管中途大关。熔体甘油果冻在水浴中于55℃进行30分钟。 </li><li>倒入一小滴甘油，约300微升，从原液到两个显微镜载玻片。使用组织，传播甘油薄拉揭掉在每个幻灯片。这提供了一个基础，可以很容易地去除甘油果冻步骤5.6。 </li><li>切尖掀起了过滤1000微升枪头。使用剪切枪头1000-1500微升的温暖甘油果冻慢慢转移到每个显微镜载玻片。请注意，以防止泡沫。 </li><li>让甘油果冻滑梯坐5分钟RT设置。 </li><li>将在60毫米×15毫米的培养皿每个甘油果冻幻灯片，并盖上保鲜膜。发生在4°CO / N。可选，存储甘油果冻幻灯片长达5天4℃。 </li></ol> 4.安装蛋钱伯斯<ol><li>使用样品从步骤2.5，吸管若干3微升的跨越一个甘油果冻滑70％甘油蛋室滴。继续移液3微升滴，直到所有的鸡蛋厅在这张幻灯片。确保每一滴含有至少十蛋室。同时，热甘油果冻的培养管在55℃水浴。 </li><li>将鸡蛋室甘油果冻滑解剖显微镜下。调整倍率以使只有一滴卵腔中的视图（ 图1B）的字段。 </li><li>用30号针头的勺子，拿起所需的阶段卵室。必要时，独立所需卵子从卵巢管链用针作为一个刀腔。 </li><li>以避免碎片可以与成像干扰，蛋室传送到第二甘油胶冻滑动，具有面向左（ 图1E -1）前。重复，直到至少有7蛋室排成一列（ 图1C）。 </li><li>直到所有所需的卵室被转移到第二甘油果冻滑形成七个新栏目。 </li><li>撕下一小块组织和扭曲的。用这个来轻轻触摸每一个从蛋室吸收多余的PBS甘油。小心不要删除蛋室。 </li><li>切断尖端的过滤200微升枪头。使用此转移甘油果冻覆盖蛋室的所有列的150微升。 注意：需要以覆盖列甘油胶冻的量是基于卵腔的阶段和列的数目。量可以进行调节。 </li><li>让装蛋室坐5分钟，在室温直到甘油胶冻的顶层完全固化（ 图1D）。 </li><li>装的鸡蛋在商会60毫米×15毫米的培养皿的地方滑动，并用保鲜膜覆盖。储存在4°CO / N，使甘油果冻层凝固在一起（的地方在黑暗中，如果有关注漂白）。 </li></ol> 5.准备蛋钱伯斯的成像<ol><li>放置装蛋室幻灯片解剖显微镜下。调整倍率以使蛋室只有一列是在视场。 </li><li>采用45°角的微型手术刀，切straigHT线沿右侧的蛋室（接近卵腔的后侧）的第一列。 （ 图1D-E） </li><li>接着，切断上述第一蛋室的顶部和下面的最后蛋室在列。在这一点上的卵腔的列应三边裁剪。 </li><li>使用microknife，沿着列的左侧切片，尽可能接近到的蛋腔（ 图1E -3）的前侧。 </li><li>吸取100微升冷1X PBT过切列。 </li><li>使用圆边刮刀除去多余的甘油胶冻周围的蛋室并丢弃切列的三侧，留在幻灯片的其余部分。 </li><li>将在蛋室前侧了晋级的锅铲和列分开初级甘油果冻块。 </li><li>准备样品或者反转（5.8.1）或直立（5.8.2）显微镜。 <ol><li>对于倒置显微镜：转让吨蛋室，他列与蛋室前侧朝下盖玻片。直到甘油果冻中的所有列已被删除，并安装重复步骤5.2-5.8。添加一对夫妇的50％PBS-甘油滴上的鸡蛋室每个块，以防止其干燥。像鸡蛋室直接盖玻片。 </li><li>对于正置显微镜：蛋室的柱转移到与前侧朝上载玻片。直到甘油果冻中的所有列已被删除，并安装在显微镜载玻片重复步骤5.2-5.9。添加一对夫妇的50％PBS-甘油滴上蛋商会和盖砖每块有＃1个半盖玻片。 </li></ol></li></ol>

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The authors have no competing financial interests.

在这里，我们描述的方法来安装和图像小，从终端的角度上制定的蛋室。成像蛋室常见的技术横向的意见进行了优化，主要允许内 - 外滤泡细胞的精确的可视化，当用荧光抗体染色。在观看多个焦平面使用的Z-堆栈或三维重建助剂，但仍有不足之处为亚细胞的椭圆卵腔室（ 图2D）的极点的分辨率。虽然这可以部分地用诸如超分辨率成像的最新的显微技术来克服，这些方法是昂贵的并且...

研究果蝇提供了巨大的见解的各种现象的遗传调控。特别是，出现了对蛋发展广泛的研究，因为卵子发生提供了一种易于处理的方式来调查许多不同的发育过程，包括组织图案化，细胞极性的变化，细胞周期的开关，和翻译调控1,2,3,4。卵子发生过程中一个重要的事件形态是一组细胞称为边缘细胞（5审查）的规格，收购蠕动，和迁移。因为细胞迁移是动物形态的一个主要特点，并且因为这个过程的基因调节是非常保守，在果蝇确定的机制很可能是在其他情况下非常重要的。因此，我们正在调查边境细胞迁移的分子控制。对于我们的研究，我们已经开发出一种新的方法来观察开发鸡蛋的前极，其中，边缘细胞产生，检查他们详细介绍了如何发展。 内卵巢，卵腔室中的不同发育阶段沿链称为卵巢管，其包封在薄鞘（ 图1A）存在。每个鸡蛋室将继续形成一个鸡蛋。在卵子发生，几种不同类型的细胞必须协调发展的方式。 D.在果蝇卵室由16个种系细胞，包括1卵母细胞，由单层滤泡上皮6包围。出现在上皮的前部和后部磁极少数特殊细胞，称为极性细胞。 6-8边缘细胞起源于卵室的前上皮细胞，诱导细胞极性5,7。在中期卵子发生（9级），边界细胞从他们的邻居和分离的滋养细胞之间迁移的卵室5,7的后到达卵母细胞。这个动作必须完成而边缘细胞停留在周边两个非能动极性细胞集群，使之成为一个类型的集体细胞迁移。边界小区集群的成功迁移确保蛋壳的珠孔，这是必要的施肥适当发展。 前极性细胞指示边境细胞命运通过激活的信号传导级联。极性细胞分泌细胞因子，不成对（UPD），其结合跨膜受体，无圆顶（DOME），对卵母细胞发育的8,9级8中相邻的毛囊细胞。 UPD的结合导致的Janus酪氨酸激酶（JAK）磷酸化转录（STAT）8,10,11的信号转导和激活。 STAT然后移动到细胞核来激活转录。慢边缘细胞（SLBO）是一种转录因子是STAT的直接转录目标，也需要边境细胞迁移12。蛋室外侧观点表明吨帽子STAT活性调节横跨前壁上皮8,11,13-阿松香三烯梯度。最接近极性细胞毛囊细胞具有活化的STAT的最高水平，因而它们变得边缘细胞和侵入相邻的种系组织。 理解边界细胞是如何内指定，并从上皮脱落，我们需要观察如何组织的组织结构。如果我们把鸡蛋室来自前上的角度来看，我们希望在周边极地细胞的毛囊细胞STAT活动的径向对称。一个端视图还更精确地之前和脱离比不同的焦平面相比较的细胞中表现出的膜蛋白和细胞间的接口的差异。因为蛋室是长方形和彼此附接由柄细胞，它们定居到载玻片横向，从而难以观察前架构。因此，有关的细胞在卵腔的磁极多的信息具有被推断，从横向的看法。虽然一些信息可以通过光的部分，光散射，光漂白算法的3-D重建，并在Z轴分辨率较差限制得到使该信息少在没有像超分辨率显微镜14昂贵的技术详细和可靠。其他种类的部分的基于成像（例如，电子显微镜或薄样切片机的切片）需要大量的操作的组织，包括脱水，增加了对工件的可能性。因此，我们开发了一种新的方法来形象D.果蝇卵室，而直立。这种方法已经被证明非常有用在阐明如何游动细胞注定（见代表性的成果和曼宁等人，正在审查 ），并且很可能是更广泛的有价值的卵子等方面的研究。

<table border="0" cellpadding="0" cellspacing="0" width="1027">
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			<td height="40" style="height:40px;width:348px;">Name of Reagent/ Equipment</td>
			<td style="width:279px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:348px;">0.1 M Potassium phosphate Buffer (KPO4 Buffer)</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Add 3.1 g of NaH2PO4&#8226;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4&#176;C.</td>
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			<td height="25" style="height:25px;width:348px;">16% Paraformaldehyde aqueous solution, EM grade</td>
			<td style="width:279px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>methanol-free to preserve GFP fluorescence</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">30G x 1/2 inch needle&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">BD305106</td>
			<td>Regular bevel</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Active Dry Yeast</td>
			<td style="width:279px;">Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten female flies</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Bovine Serum Albumin</td>
			<td style="width:279px;">PAA-cell culture company</td>
			<td style="width:119px;">A15-701</td>
			<td>Used in NP40 Wash Buffer</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Dumont #5 Forceps</td>
			<td style="width:279px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td style="width:283px;">Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:283px;">5mg/ml stock solution&#160;&#160;</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:283px;">Added to supplement dissection medium (10%)&#160;</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:348px;">Glass culture tube</td>
			<td style="width:279px;">VWR</td>
			<td>47729-576</td>
			<td>14mL</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glass Depression Slides</td>
			<td style="width:279px;">VWR</td>
			<td>470019-020</td>
			<td>1.2 mm Thick, Double Cavity</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glycerin Jelly&#160;</td>
			<td style="width:279px;">Electron Microsocpy Sciences</td>
			<td>17998-10</td>
			<td>Mounting media</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glycerol</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB15760</td>
			<td>70% in PBS</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">IGEPAL CA-630</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">I3021-500ML</td>
			<td style="width:283px;">(interchangable for Nonidet P-40)&#160; Used in NP40 Wash Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica Fluorescent Stereoscope&#160;</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica SP5 Confocal Microscope</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>40x/0.55NA dry objective&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Micro spatula&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">82027-518</td>
			<td>Stainless steel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microknife&#160;</td>
			<td style="width:279px;">Roboz Surgical Instrument Company</td>
			<td style="width:119px;">37-7546</td>
			<td>45&#7506; angle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microscope Slide</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">16004-368</td>
			<td>75x25x1 mm</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:348px;">NP40 Wash Buffer</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:279px;">Life Technologies</td>
			<td>10378-016</td>
			<td>Added to supplement dissection medium (0.6X)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Petridish- Polysterine, sterile</td>
			<td style="width:279px;">VWR</td>
			<td>82050-548</td>
			<td>60Wx15H mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Phosphate Buffer Saline</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3813-10PAK</td>
			<td>10 packs of Powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate dibasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3786-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate monobasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Schneider&#8217;s Insect Medium</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;"> 
			11720018</td>
			<td>With L-glutamine and sodium bicarbonate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Sodium azide</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td>Used in fluorescent antibody staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Sodium chloride</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S3014-500G</td>
			<td>Used in NP40 Wash Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">TRIS-HCL</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB70144</td>
			<td>1M TRIS-HCL, pH 7.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Volocity 3D Image Analysis Software</td>
			<td style="width:279px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>For processing confocal Z-stacks</td>
		</tr>
	</tbody>
</table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

直立成像方法使我们能够看到细胞中的前滤泡上皮的直接的组织在阶段8，一种一般标记毛囊细胞命运，眼中的缺席（EYA）蛋白质，以及核DNA的DAPI标记，表明即使表达横跨细胞的这个领域，并表明，所有的细胞可被视为具有类似染色强度（ 图2B“）。蛋白响应于细胞因子UPD调节，但是，显示出可变图案和表达水平。极地细胞释放UPD顶部之前，这个阶段卵子发育16,17的，所以我?...

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.5K 次观看.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.