这项工作是由全国环境卫生科学研究所的支持K22ES012264 AJRB]和美国癌症协会InstitutionalResearch格兰特[ACS - IRG - 00 - 173 - 04]试点projectaward [AJRB]。我们也感谢批判性阅读的稿件和评论，尤其是视频和亚当布朗主教实验室的成员，什么不该做的例子。我们感谢让我们解剖视频拍摄他解剖的范围/摄像机设置使用Greehey儿童癌症研究所的博士唐纳德麦克尤恩。我们感谢达龙布朗科尔特仪器锐化和修复我们的显微切割工具。

1。删除多余的组织，从外面的眼睛<ol><li>一个35mm的培养皿的盖子，倒入1X PBS。水平应略低于盖子的唇。 </li><li>直钳，转让眼（S）的存储管使用的35mm盘。眼睛被淹没在PBS的原因有两个：1。多余的组织将“漂浮”，从眼睛，这样​​你可以看到轻松删除它们，和2。在清扫，眼睛自然会保持球形，而处于悬浮状态，让您的工作而不是反对这种形状。 </li><li>都对使用直角钳（45 °和15 °），轻轻取出尽可能多的肌肉和结缔组织，拉动对粮食的组织（即向着角膜）。一些结缔组织将保持不变。应作出努力，以尽可能少的挤压眼球。 </li><li>春天剪刀贸易15 °钳。按住眼睛的稳定与45 °钳，使用弹簧剪刀剪去余下的肌肉和结缔组织，以及视神经。 *提示：使用外边缘的剪刀，以推动对粮食组织（朝corneo巩膜鸿沟），然后修剪。继续，直到所有多余的组织已经从眼睛中删除。 </li></ol> 2。取下的角膜和晶状体<ol><li>角膜朝上，使用45 °钳，捏在角膜中心的小褶皱。 </li><li>使用Spring的剪刀，让一个小切口，在皮瓣的基础。让我们去的皮瓣。 </li><li>控股的眼睛仍然与您的镊子，剪刀刀片插入较低的和垂直的切口，并削减向corneo巩膜鸿沟。尽量使你的下刀片，所以下的虹膜角膜和虹膜切开。不要削减一路corneo巩膜鸿沟。保持剪刀插在眼睛上。 </li><li>使用45 °钳，小心地转动眼睛，使你的剪刀平行corneo巩膜鸿沟，使一小截。慢慢转动眼球360 °，使小切口周围所有的方式。再次，尽量保持较低的弹簧剪刀刀片，使下虹膜虹膜和角膜切开。电梯的虹膜和角膜离眼睛休息和丢弃。 </li><li>在其一侧倾斜的眼睛和对眼睛的后半部轻轻下压，迫使镜头。丢弃的镜头。剩下的眼罩内两旁是由神经视网膜，这看起来应该光滑，不透明。 </li></ol> 3。季眼罩，造成4 - “petaled”，花状结构<ol><li>眼睛应定位眼罩开幕是你的身体垂直。使用45 °钳稳定和位置眼罩。 </li><li>在你面前的弹簧剪刀，所以他们是垂直你的身体中，将其插入顶边开放眼罩弹簧剪刀的刀片。使用45 °钳，小心地将眼罩，使一个单一的，直切可走向视神经头corneo巩膜鸿沟。所有伤口应垂直corneo巩膜的鸿沟，除非指定的，应该没有到它切割尽可能靠近视神经头的。 </li><li>眼罩180 °旋转，与你的身体保持垂直。在步骤3.2中使用的技术，进行第二次垂直切割。 </li><li>旋转眼罩和重复3.2的技术，使两个多削减。所有四个削减应尽可能接近90 °尽可能。请记住，映入眼帘的是不完美的球形，因此会有一些变异。 </li></ol> 4。切成两半的四个“花瓣”，在八个花瓣状结构<ol><li>将驻扎眼罩，从35毫米盘到一个干净的幻灯片。朝上的眼罩，舀下45 °钳。小心地将其取出液体放在一个干净的幻灯片。 </li><li>使用直角钳都对，轻轻打开了眼，神经视网膜朝上。不删除神经视网膜它有助于眼睛保持球形，这样你就不会反对。它还在以下过程中被意外损坏保护视网膜色素上皮。 </li><li>旋转滑动，直到花瓣之一是你的身体垂直。 </li><li>使用15 °钳，轻轻抓住花瓣的角落。花瓣的角落应角膜，神经视网膜或视网膜色素上皮。角落里轻轻抬起，允许它保留其曲率。 </li><li>插入底部的刀片之间的花瓣和幻灯片的春天剪刀。行了你的剪刀等，可以从一个单一的，直线，垂直切割corneo巩膜鸿沟走向视神经头。削减非常接近，尽可能的withou视神经头吨削减到它。让花瓣，允许它在幻灯片上的谎言。 </li><li>旋转滑动90 °，使未来的花瓣是你的身体垂直。重复步骤4.5。 </li><li>重复步骤4.5和4.6两次以上。由此产生的结构应该看起来像一个八瓣花。 </li></ol> 5。取出视网膜神经<ol><li>移液器100-200μL的1X PBS到标本。这将防止从坚持为你的视网膜色素上皮剥离的视网膜神经。 </li><li>视网膜神经通常是松散连接的RPE。但是，它采取的做法和技能，以去除神经视网膜的RPE。轻轻抓住直角钳一对视网膜神经花瓣，同时稳定（按住），直角钳的另一对眼睛休息。将视网膜神经远离眼睛休息。继续此过程，直到整个神经视网膜已被删除。 *提示：坚持到视神经头15 °钳尖锚眼睛。然后用45 °钳扫下方视网膜神经和抬离和距离。它通常是最好的解除神经视网膜视神经头走向corneo巩膜鸿沟。可以肯定的，绝尘而去的虹膜任何松动的残余。虹膜是很粘，否则可以卡住的RPE。 </li><li>用1X PBS冲洗标本。移液器100-200μL的1X PBS标本和吸备份。丢弃的PBS。必要时重复来清除灰尘和碎片。 </li></ol> 6。你在幻灯片装入标本（S） <ol><li>吸取80-90μL，90％的甘油长方形到一个新的幻灯片。使用枪头方矩形均匀，甘油涂抹。 （这是一张幻灯片上安装两个视网膜色素上皮的过程。） </li><li>插入的下臂下的标本15 °钳。轻轻抬离标本解剖幻灯片。 </li><li>缓慢地降低到甘油的标本。轻轻摆动的钳为你拉他们从下标本。 </li><li>重复步骤6.1-6.3其它标本。 </li><li>旋转滑动，使长边垂直于你的身体。按住玻璃盖在一个45度角到幻灯片，这是动人的幻灯片。走向甘油涂抹，直到他们接触。玻璃盖和幻灯片之间保持联系，慢慢地移动远离涂抹的玻璃盖，走向边缘的幻灯片。最多的幻灯片的长边和玻璃盖，使他们是平行的。握住你的拇指和食指之间的玻璃盖的四角。 </li><li>在另一只手拿起15 °钳。摇篮上（免费）边在玻璃盖的臂弯。很慢，低的玻璃盖。当接触之间的玻璃盖和甘油，甘油迁移停止并等待。重复此降低，并等待，直到RPE整个坐骑完全覆盖。这是为了防止从捕捉气泡的高粘度的甘油。不推盖玻片 - 甘油会挤压下，使得附着力很差。 </li><li>有了明确的指甲油，油漆各地的外围玻璃盖和幻灯片的满足。 </li><li>放置在一个空架干之上的幻灯片。 </li></ol> 7。代表性的成果： 此过程的结果应该是一个结构，看起来像一朵花，应该是相当对称。 <img src="/files/ftp_upload/2563/2563fig1.jpg" alt="图1" /> 图1。整个安装的RPE从一个野生型C67Bl/6J鼠标 ，从黑色或刺豚鼠动物的视网膜色素上皮应该是深褐色，并应具有光滑的表面。花瓣的地形起伏是正常的通知。对任何给定的标本的色素沉着可能会有所变化，由于变密度的RPE和底层脉络膜的色素沉着。白色箭头指向色素减退的“渠道” - 这是正常的是，由于眼睛的基本血管。蓝色箭头指向物理损坏的视网膜色素上皮和基本脉络。 <img src="/files/ftp_upload/2563/2563fig2.jpg" alt="图2" /> 图2。整装稀鼠标的RPE。收获稀动物标本的范围可以从几乎透明的颜色的Cafe au Lait和任何一个标本是想在它的可变性，在这里的黑色圆圈表示。在一般情况下，从年幼的动物收获的视网膜色素上皮的重量更轻，从年纪较大的动物收获是较深。黑色箭头指示的一些底层的血管，出现色素减退。 <img src="/files/ftp_upload/2563/2563fig3.jpg" alt="图3" /> 图3。鬼笔环肽染色可以用来检测身体损害的RPE。鬼笔环肽染色清晰地勾勒出的细胞膜，上皮六角形。 （一）例如，从一个黑色的鼠标。 （二）例如，从稀鼠标。 <img src="/files/ftp_upload/2563/2563fig4.jpg" alt="图4" /> 图4。从稀释的小鼠解剖不当的视网膜色素上皮 （A）括号表示角膜的边缘太宽，这可能会导致屈曲和/或折叠。在黑色圆圈中，你可以看到尚未从后面的眼睛中删除的多余组织的过度。他们尤为明显，因为样本是从稀动物。左下角的花瓣是部分折叠。 （b）本整装风车的外观。黑色箭头表示一些削减作了。 corneo巩膜鸿沟走向视神经头的削减，很多都不是直径。 （三）如何削减已取得的直径和垂直切线，黑色箭头表示。

<ol>
	<li>Bishop, A. J., Kosaras, B., Sidman, R. L., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzo(a)pyrene+andX-rays+induce+reversions+of+the+pink-eyed+unstable+mutation+in+the+retinal+pigmentepithelium+of+mice.">Benzo(a)pyrene andX-rays induce reversions of the pink-eyed unstable mutation in the retinal pigmentepithelium of mice.</a> Mutat. Res.. 457, 31-31 (2000).</li><li>Reliene, R. H. l. a. v. a. c. o. v. e., Mahadevan, A., Baird, B., M, W., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diesel+exhaust+particles+cause+increased+levels+of+DNA+deletions+after+transplacental+exposure+in+mice.">Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice.</a> Mutat. Res. 570, 245-2452 (2005).</li><li>Bishop, A. J., Barlow, C., Wynshaw-Boris, A. J., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm+deficiency+causes+increased+frequency+of+intrachromosomal+homologous+recombination+in+mice.">Atm deficiency causes increased frequency of intrachromosomal homologous recombination in mice.</a> Cancer Res. 60, 395-399 (2000).</li><li>Brown, A. D., Claybon, A. B., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+WRN+helicase+domain+is+not+required+for+spontaneous+homologous+recombination-mediated+DNA+deletion.">Mouse WRN helicase domain is not required for spontaneous homologous recombination-mediated DNA deletion.</a> J. Nucleic Acids. , (2010).</li><li>Claybon, A., Karia, B., Bruce, C., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP1+suppresses+homologous+recombination+events+in+mice+in+vivo.">PARP1 suppresses homologous recombination events in mice in vivo.</a> Nucleic Acids Res. , (2010).</li><li>Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53-,+and+Gadd45a-deficient+mice+show+an+increased+frequency+ofhomologous+recombination+at+different+stages+during+development.">p53-, and Gadd45a-deficient mice show an increased frequency ofhomologous recombination at different stages during development.</a> Cancer Res. 63, 5335-5343 (2003).</li><li>Bodenstein, L., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+development+of+the+mouse+retinalpigment+epithelium.+Part+I.+Cell+and+tissue+morphometrics+and+topography+of+mitoticactivity.">Growth and development of the mouse retinalpigment epithelium. Part I. Cell and tissue morphometrics and topography of mitoticactivity.</a> Dev Biol. 121, 192-204 (1987).</li><li>Burke, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+phenotype+and+the+RPE:+is+the+answer+blowing+in+the+Wnt?.">Epithelial phenotype and the RPE: is the answer blowing in the Wnt?.</a> Prog Retin. Eye Res. 27, 579-595 (2008).</li></ol>

视网膜色素上皮是联合国的 P检测的站点，在体内的同源性定向修复检测。 联合国的 p检测已用于研究不同的DNA损伤的影响 ，1,2和频率上的同源性定向修复DNA损伤信号和修复基因3,4,5。此实验是非常敏感的检测单细胞活动的RPE 1。它也可以检测到 6在开发过程中的同源性定向修复事件的时间差异。鼠眼径向向外发展视神经7，<...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>straight forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-4903</td>
<td>tip: .08 x .04 mm material: INOX</td>
</tr>
<tr>
<td>45&deg; forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5005</td>
<td>tip: .05 x.01 mm material: INOX</td>
</tr>
<tr>
<td>15&deg; "up" forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5045A</td>
<td>tip: .1 x.06 mm material: INOX</td>
</tr>
<tr>
<td>spring scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5604</td>
<td>comb. tip width 0.3mm cutting edge length 3mm material: stainless steel</td>
</tr>
<tr>
<td>binocular dissecting microscope</td>
<td>&nbsp;</td>
<td>Ziess</td>
<td>Discovery V.8</td>
<td>use reflected light source</td>
</tr>
<tr>
<td>phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A22283</td>
<td>Alexa Fluor 546</td>
</tr>		</tbody>
		</table>
		</div>

dissection of a mouse eye for a whole mount of the retinal pigment epithelium

视网膜色素上皮（RPE）是哺乳动物的眼睛后面，刚下的视网膜神经元，其中包含的光感受器（杆和视锥细胞）。色素上皮是单层联营密切的正上方的神经视网膜色素立方细胞和。这种关联使视网膜色素上皮对视网膜疾病的研究极大兴趣。的RPE也是一个同源DNA修复， 联合国的 P检测体内检测站点。鼠标的眼睛是特别困难的解剖，由于其体积小（直径约3.5毫米）和球形形状。本文演示了详细的剥离导致眼睛在整个视网膜色素上皮安装程序。在此过程中，我们将展示如何工作对眼睛的球形结构，而不是的。简言之，结缔组织，肌肉，和视神经从眼球后部。然后，角膜和晶状体将被删除。下一步，战略裁员，在重大其余组织的扁平化的结果。最后，视网膜神经轻轻地升空，揭示了一个完整的视网膜色素上皮，这仍然是连接到底层脉络膜和巩膜。这整个挂载可用于执行联合国的 P检测或免疫组化或免疫视网膜色素上皮组织的评估。

Watch this Scientific Journal Video about Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium at JoVE.com

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

解剖一只老鼠眼的一个正式的示范，导致在整个视网膜色素上皮的坐骑。

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

dissection-mouse-eye-for-whole-mount-retinal-pigment

Research

JoVE Journal

Neuroscience

49.1K 次观看.  Greehey Children's Cancer Research Institute and Department of Cellular and Structural Biology. 解剖一只老鼠眼的一个正式的示范，导致在整个视网膜色素上皮的坐骑。

视频: Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

使用清晰组织清除方法对全山视网膜进行免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

用于研究视网膜神经节细胞的小鼠视网膜分离和甲醇处理

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

作者聚焦：用于研究视网膜神经节细胞的基于甲醇的整装制剂  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

基于甲醇的全支架制备用于视网膜神经节细胞的研究

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

Methods Article

解剖鼠标眼的视网膜色素上皮的全山