Igal Gery and David Hicks for anti-SAG antibodies. Ram Fridlich for reading the manuscript.

注：该程序是经伦理委员会达尔文大学的皮埃尔和玛丽·居里（CE5 / 2009/048） 1.准备明胶溶液，仪器的vibratome，文化传媒与文化板块<ol><li>准备至少一天实验前20％的明胶溶液。 <ol><li>引擎盖下，加入500μl庆大霉素[10毫克/毫升]中，以500毫升一瓶含CO 2独立的媒体（CO 2 -i）。 </li><li>在一个单独的烧杯中，在95℃的加热块上添加20毫升二氧化碳-I的（从步骤1.1.1）和加热并不断搅拌15分钟。观察的颜色变化到黄色，表明溶液已准备好以供进一步使用。 </li><li>权衡4克明胶和逐渐差具有增加的搅拌和加热上述制备的溶液（ 步骤1.1.2）在95℃下45分钟，以得到黄色溶液。允许明胶溶液下冷却罩为大约5分钟。 </li><li>倒入4毫升明胶溶液放入直径35 mm的培养皿中没有引入气泡。保持培养皿在21℃30分钟。盖上塑料薄膜的菜肴，把菜肴倒挂。任选地，保存菜肴，在4℃下使用前长达2个月。 </li></ol></li><li>制备4％的明胶溶液。 <ol><li>引擎盖下，在42℃的加热块上添加25毫升的CO 2 -i介质中的无菌的塑料容器，和热。从加热块中取出介质，并逐渐加入1g明胶到热介质，搅拌。快速返回容器中的加热块（42℃）上，并在此过程中保持温暖。 </li></ol></li><li>设置-起来的vibratome设备： <ol><li>删除包含储存于4℃的20％的明胶溶液中的菜肴。切断与手术刀明胶。翻转明胶切片，贴在的vibratome的使用一滴s的黑盘支持UPER胶水。 </li><li>打破刀片成两半，并插入一个半到的vibratome控股插座。将黑色的支持光盘上的vibratome设备。打开黑色旋钮右侧。固定在所述的vibratome的头部的保持插座。加的CO 2 -i介质足够量中的vibratome罐以覆盖明胶块。 </li><li>接通的vibratome切成阿胶块三个100微米片。继续进一步使用的阿胶块。 </li></ol></li><li>制备40毫升培养基： <ol><li>罩下制备的Dulbecco改良的Eagle培养基（DMEM）胎牛血清（FCS）的10％。制备1mg / ml的聚-D-赖氨酸用磷酸盐缓冲盐水（PBS）pH 7.4的。 </li><li>在一个96孔板中，添加聚-D-赖氨酸的2微克/厘米2涂覆的孔的底部。在5％的CO 2培养箱中孵育该板45分钟，在37℃。孵育后，除去PBS，并替换为200μl的/孔的DMEM和地点再次在37℃下在5％CO 2培养箱中在使用前。 </li></ol></li></ol> 2.解剖整个视网膜的<ol><li>剜除鼠标： <ol><li>根据欧洲指令六十三分之二千〇一十牺牲小鼠：通过颈脱位。切头弯剪刀，并将其放置在一个直径100毫米的培养皿经消毒眼用消毒后。 </li><li>去除眼部受切片非常仔细地使用弯曲的抓地力后，已经脱离了视神经​​。然后把双眼含无菌CO 2 -i培养基21°C 35毫米的培养皿。 </li></ol></li><li>取出视网膜： <ol><li>使一个孔在眼睛肢用18G的针的帮助下的水平，以便能够引入剪刀到眼睛的地球。介绍孔内直剪刀小心切割下虹膜，巩膜上眼球的所有周边删除睫状体。 </li><li&gt;取下角膜和透镜，并保持在眼睛的后房与视网膜邻接的巩膜。有两个细交手，除去位于后室内的玻璃没有损坏视网膜。执行四个径向切口（视网膜/巩膜），以允许在视网膜的平坦化。 </li><li>好转眼球和剥离它作为一个“橙色”。分离从巩膜和视网膜色素上皮细胞（RPE）的视网膜。卸下仍连接在视神经的水平非常仔细地用细弯剪刀视网膜。 </li><li>分离从朝视网膜的具有两个细把持中心的周边的其余玻璃体。确保所有玻璃体完全除去以使视网膜的扁平化。 </li><li>切割塑料吸管的末端和视网膜转移到含有CO 2 -i介质与塑料吸管35毫米直径的培养皿。 </li></ol></li></ol> 3.密封平mounte的ð视网膜上阿胶块<ol><li>从的vibratome罐（阿胶块是免费的）30毫升CO 2 -i介质允许平板式视网膜密封-删除20。 </li><li>用塑料吸管视网膜转移到载玻片上并转移到明胶片朝下明胶片上的感光体前2 -i介质加一滴的CO。 </li><li>通过轻轻地注入在视网膜和明胶块之间的块的一侧温热4％明胶和同时在另一侧排出暖和明胶附着在视网膜的明胶块。 </li><li>抽吸4％明胶用巴斯德吸管。等待10分钟，以允许总的密封。加入CO 2 -i中沉浸块，完全的刀片。 </li></ol> 4.切片视网膜注：该步骤是关键的，得到层感光细胞没有其他视网膜细胞（ 图1）。 <ol><li>从明胶块的顶部开始，切割100微米的连续切片到达视网膜。然后切100 - 内层120微米的部分。根据不同的应用，该层可能会被丢弃或保存在液氮中。确保的vibratome的速度很慢（在1或2）内层或光感受器的层的切片中。 </li><li>执行对应于所述内和外视网膜之间的界面处的视网膜的中间体15微米的部分。遵守本节在显微镜下。缺乏血管表明外视网膜已经达到。 </li><li>切割200微米的外视网膜（感光层）与明胶，并将其转移到充满用3ml的CO 2 -i介质的35毫米直径的培养皿中。保持它在冰上，直到所有的视网膜的所有感光层段获得。 </li></ol> 5.进行分离感光细胞<ol><li>就拿菜（ES）含有感光层（多个），并把它（它们）的培养箱，在37℃下进行10分钟。 </li><li>利用镊子轻轻地从明胶分离感光层和在一个新的培养皿填充用3毫升林格氏溶液传送。重复每个感光层准备这一步（5.2）。 </li><li>孵育2个单位的木瓜蛋白酶的用25微升的活化剂溶液（1.1毫摩尔EDTA; 5.5毫L-半胱氨酸; 60μMβ巯基乙醇）在5ml的聚丙烯无菌管中并在5％CO 2培养箱孵育它30分钟，在37℃下。木瓜蛋白酶的激活是在37℃下实现的。在此孵育后，切断感光层分为2 mm 2的片（ 未太小 ），并且这些片转移到5ml的试管中。 </li><li>用1.5ml的溶液林格氏溶液，随后通过重力沉降漂洗视网膜两次。除去从与试样管的所有剩余林格氏液。 </li><li>添加475微升林格氏液含有活化木瓜蛋白酶和混合管。添加这种溶液到含有视网膜管。 </li><li>孵育视网膜20分钟的管中在37℃在5％CO 2的培养箱中培养。停止加入在DMEM1毫升10％FCS中的反应。添加脱氧核糖核酸酶25 U I（DNA酶I），以消化DNA从死亡细胞。使用1毫升移液管小心地均化的细胞悬浮液。旋转，在50×g离心6分钟，在室温。 </li><li>弃去上清液，以除去血清的痕迹，并与补充添加DMEM培养基（见表的具体reagents_equipment）到细胞沉淀，并仔细重悬细胞悬液与1毫升吸移管。旋转，在50×g离心6分钟，在室温。重复此步骤（5.7）一次。 </li></ol> 6培养感光细胞<ol><li>的小鼠在PN8所述感光层含有用这种技术大约1至1.5百万个细胞。种子光感受器细胞以1×10 5个细胞/ cm 2在培养板上用培养基和培养在5％CO 2培养箱将细胞5天，在37℃。在第5天，进行免疫化学和通过抗体的供应商提供的免疫印迹研究以下方法。 注：步骤停止文化和初步测试是在参考21描述。 </li></ol>

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The authors have nothing to disclose.

视网膜是在生物学模型的器官。研究视网膜导致6生物学重大发现。它是在第一个肿瘤抑制基因RB1的起源。它揭示了通过与sevenless之子相互作用的受体酪氨酸激酶和MAP激酶分子之间的联系。它参与了PAX6，第一主控制基因器官形态发生的发现。它是在中心补体因子H（CFH）的遗传关联的与年龄相关性黄斑变性（AMD），确定全基因组关联屏幕（GWAS）第一疾病易感基因。最后，它导致了第一个成功的基因治疗莱伯先天性黑朦，第一矫正基因治疗试验中的任何人的遗传性疾病。这个机构的结构是保守的，在大多数脊椎动物物种。其可用于操作在体内已初鼓励对功能基因组学研究的一分钱这种不可分割的一部分RAL神经系统。 我们在这里展示了如何将感光体层从视网膜的内层由的vibratome切片分离。这个步骤是关键的，得到纯的感光体培养物。我们解剖协议使得污染RPE细胞不太可能。 其中一个主要的挑战是视网膜所必需的部分适当的内和外视网膜的扁平化。视网膜切片最好在视网膜与小直径的那些从啮齿动物和该直径是与目前可用的材料的技术中的一个限制。 它是在开始一个生物学意义的实验中，以实践在视网膜上的一个样品之前为宜。我们通过从培养的感光细胞得到的代表性结果示的方法中，使用的材料，以同时执行的mRNA和蛋白质的表达的研究。表达研究也可以在sectio备选地进行通过激光捕获显微切割获得纳秒，但文化是最好的执行使用的vibratome切片。我们可以使用显微激光技术具有完整的不同的策略。但是，以收集在感光层与培养的显微切割装置中，这将是必要的，以避免固定剂，而这将显著复杂化目前的方法。 我们正在开发旨在感光的视网膜色素变性的车型变性过程中研究基因和蛋白表达的vibratome部分动力学的协议。我们认为，该协议的详细描述将是有益的研究人员在视网膜生物学领域，最特别是对蛋白质组学和代谢组学研究。

视网膜是中枢神经系统，其具有脊椎动物中保守的结构的一个组成部分。神经视网膜的神经元组织中的层，与最遥远的入射光，与视网膜色素上皮细胞（RPE）在眼睛后部紧密接触的感光体层。杆和视锥photorecptors是依靠敏感的视蛋白分子捕获光子的光敏感细胞。这些分子被封闭在一个蜂窝结构的磁盘膜，位于指向在RPE中1的方向上的光感受器的外段。这种结构，这是最常见的受光感受器变性案件很早，被更新在每天10％的税率。所谓的内层包含大部分计算从光感受器，双极，无长突和水平细胞以及神经节细胞接收到的信号中的其它神经元。后者与它们的轴突形成一个光束是视神经。这个分层是如此保守生物学家已经使用的术语移动无长突细胞，当细胞内网状层2外发现。神经元的层分布的径向缪勒胶质细胞的电枢内。双极细胞光感受器链接到神经节细胞。它们位于所述外部网状层和内丛状层之间。神经节细胞形成与双极细胞连接的内网层。所述amacrin细胞被命名为位于双极细胞和神经节细胞之间的内网层关联的细胞。外丛状层包含水平细胞。中枢神经系统的神经元层的这种独特的设置允许从内细胞层的感光体层的分离通过使用振动切片平面式视网膜。 最初，这种技术被用于分离光感受器为TRAN同种异体在RD1鼠标，人类视网膜色素变性 （RP）3模型的眼球。的RD1小鼠携带在PDE6B基因，其编码用于杆特异性磷酸二酯酶β亚基隐性突变。这个基因导致RP在人类4隐性突变。后视杆细胞已经退化，患者失去夜视，并且令人惊奇地锥光感受器，其不表达突变基因，简并为一个第二步骤。因为锥体所需的色觉和视力，患者变得逐步失明和一种有效的治疗该疾病尚未制定。通过从野生型小鼠移植感光层宿主小鼠的圆锥变性延迟3,5。由移植因为杆和双极细胞之间的突触连接只能在r的一个特定阶段可以得到在杆锥退行性模型丢失杆不能被替换etinal发展，其标志是NRL表达式6的发作。光感受器的层是通过外科手术，在无杆RD1视网膜的视网膜下空间引入，视网膜色素上皮和对应于只有3％的剩余的感光体，所述锥体的外视网膜之间。手术后两周，从移植了一个正常的感光层中的动物的视锥细胞的40％存活相比，移植了内视网膜细胞，或者假手术动物的正常层的动物。锥存活的地形，散开的过度突变的视网膜位于移植组织的位置的整个表面上指示的保护作用是由于扩散分子7。 下一步，我们使用了共培养系统以及调节的培养基，以验证保护作用依赖于蛋白的杆8,9的分泌的事实。我们推测，这种蛋白质会被EXPR在疾病的第一阶段由杆和它们的死亡连续且具体地说ESSED将触发次级锥形变性由保护信号从杆在非细胞自主方式10中的损失。由于锥介导的中央视力在灵长类动物的重要性，这个假定的蛋白质将是一个很强的针对性治疗手段的RP。在保存RP视锥细胞在理论上阻止共150万世界各地的患者，成为盲人11。我们使用了高内涵筛选方法和锥丰富的文化模型来确定的cDNA从视网膜cDNA文库编码12杆衍生锥活力因子（RdCVF）。 RdCVF是NXNL1基因，有趣的是同源的对参与氧化还原平衡13硫氧还蛋白的蛋白质编码基因的剪接产物。该基因的第二拼接产物，RdCVFL是保护它的目标，对氧化D中的τ蛋白的酶豪悦国际14。 RdCVF的施用防止锥的仲变性和其视觉功能在一个隐性的损失，以及反相12,15主导模式。这表明这种创新的治疗策略16的两个重要方面。首先，它可以在大多数的情况下，RP应用于在一个基因无关的方式。其次，违背了竞争因素CNTF，RdCVF生存与视觉功能17的维护相关。没有功能效果可以解释缺乏CNTF的管理，临床受益RP患者18的原 ​​因。 RdCVF是最有可能的，因为在体外锥体救援由RdCVF免疫耗竭12抑制杆和视锥之间的重要的生存信号中的一个。此外，杆衍生锥体 ​​生存能力基因的破坏导致光感受器功能障碍和易感性氧化应激19。 使用感光层是在参与神经变性疾病20一种新颖的氧化还原信号的识别RdCVF的起源和。这个手稿描述了用于分离和从感光层培养细胞RdCVF活性表征的协议。该感光体可保持在培养5至7天21。这种技术也可以用来研究的感光体特异基因的表达。

<table border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
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			<td height="21" style="height:21px;width:319px;"><a name="RANGE!A1:D28">Name of Material / Equipment</a></td>
			<td style="width:167px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
			<td style="width:368px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5&#8242;-diphosphocholin*</td>
			<td>Sigma-Aldrich</td>
			<td>C0256</td>
			<td>4.7 &#181;M Cytidine 5&#8242;-diphosphocholin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5&#8242;-diphosphoethanolamine*</td>
			<td>Sigma-Aldrich</td>
			<td>C0456</td>
			<td>2.7 &#181;M Cytidine 5&#8242;-diphosphoethanolamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Linole&#239;c acid/ bovine serum albumin (BSA)*</td>
			<td>Sigma-Aldrich</td>
			<td>L8384</td>
			<td>100 &#181;g/ml linole&#239;c acid/ bovine serum albumin (BSA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodo-L-thyronine*</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td>0.03 &#181;M Triiodo-L-thyronine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-wells plate</td>
			<td>Greiner bio-one</td>
			<td>655-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binocular microscope</td>
			<td>Leica</td>
			<td>MZ-75</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CO2 independent (CO2-i)</td>
			<td>Life Technologies</td>
			<td>18045054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps n&#176;5 Dumont</td>
			<td>Bionic</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from porcin skin type A</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneChip&#160;</td>
			<td>Affymetrix</td>
			<td>U74v2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin solution</td>
			<td>Life Technologies</td>
			<td>15710049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortison*</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>0.55 &#181;M hydrocortison</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin* (I)</td>
			<td>Sigma-Aldrich</td>
			<td>I1884 (ITS)</td>
			<td>0.86 &#181;M insulin (I)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain</td>
			<td>Worthington-biochem</td>
			<td>WOLS03124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P-6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone*</td>
			<td>Sigma-Aldrich</td>
			<td>P7556&#160;</td>
			<td>2.0 &#181;M progesterone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin*</td>
			<td>Sigma-Aldrich</td>
			<td>P5172</td>
			<td>0.28 &#181;M prostaglandin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrescine*</td>
			<td>Sigma-Aldrich</td>
			<td>P5780</td>
			<td>&#160;182 &#181;M putrescine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Albion</td>
			<td>EMS</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissor&#160;</td>
			<td>Moria</td>
			<td>15396-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate*</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td>1 mM sodium pyruvate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Selenite* (S)</td>
			<td>Sigma-Aldrich</td>
			<td>I1884 (ITS)</td>
			<td>0.29 &#181;M Na2SeO3&#160;&#160; (S)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine*</td>
			<td>Sigma-Aldrich</td>
			<td>T8691</td>
			<td>3 mM taurine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferrin* (T)</td>
			<td>Sigma-Aldrich</td>
			<td>I1884 (ITS)</td>
			<td>0.07 &#181;M transferrin (T)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome apparatus</td>
			<td>Leica</td>
			<td>VT1000-S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">* Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

vibratome sectioning mouse retina to prepare photoreceptor cultures

视网膜是已经从感光体组织结构，与神经元的层，这两个杆和视锥细胞在接触与视网膜色素上皮细胞在视网膜上考虑的光的方向上的最远部分的中枢神经系统的一部分，并且神经节细胞在最近的距离。这种结构允许感光层的隔离用的vibratome切片。的年龄8天小鼠解剖神经视网膜是平嵌入在4％的明胶上的20％的明胶感光层的片朝下的顶部。使用振动和双刃刀片，所述100μm厚的内层视网膜切片。此部分包含神经节细胞和内层与特别是双极细胞。为15微米的中间部分被丢弃200微米含有光感受器的外视网膜的回收之前。明胶是通过加热在37℃下除去。外层的作品是incuba泰德在500μl林格氏用2单位活化木瓜蛋白酶的20分钟的溶液，于37℃。该反应通过在Dulbecco氏改良Eagle培养基（DMEM）中加入500微升10％胎牛血清（FCS）的停止时，则25个单位DNA酶I的离心之前，在室温下加入，洗涤数次以除去血清和将细胞重新悬浮于500微升DMEM中，接种1×10 5个细胞/ cm 2。细胞生长至5天的体外和使用活/死测定其生存能力评分。培养物的纯度，首先通过显微镜观察在实验过程中确定的。纯度然后通过播种和上一个组织学载玻片固定的细胞和使用的兔多克隆抗SAG，感光体标记和小鼠单克隆抗RHO，一个视杆特异性标记分析验证。可选择地，感光体层（97％棒）可用于基因或蛋白质表达的分析和用于移植。

除了 ​​移植，在感光层也已被用于通过种子细胞用于制备感光体培养12,22研究细胞信号传导。此外，它们被用于研究基因表达和昼夜节律23,24。我们使用从野生型小鼠的细胞感光层8产后天以制备在没有FCS的感光体培养和维持细胞5天，于37℃在培养箱中5％的CO 2（ 图1E ）。然后将细胞用4％多聚甲醛固定，然后继续使用由抗体供应商或者与小鼠单克隆抗RHO提供的方法中免疫细胞化学分析（1：250，Millipore公司Mab5316）或兔多克隆抗SAG（1：200，大方有约色格里和大卫·希克斯）的礼物。我们使用抗RHO和抗SAG（虽然抗SAG标签锥体和杆），由于这样的事实，抗SAG是较早的标记，比反RHO。 CEL的比例LS阳性抗RHO抗体是比那些阳性抗SAG抗体（ 图2）低。这可以通过以下事实SAG，杆休止也由锥体这使得感光体的3％，在分离的层中，或可替代地由视网膜的产后成熟期间，凹陷的表达先于该事实表示进行说明的RHO 25,26。 在感光层也使用年龄35天核糖核酸用氯化铯超速离心27制备和杂交小鼠DNA芯片阵列野生型视网膜动物和大脑的光感受器来监测基因的特异性表达。信使对于视紫红质（RHO），S-休止（ 凹陷 ）和锥形转导（Gnat2）是专门在视网膜相比，大脑（ 图3A）来表示。的表达是在所述感光层（PR）比整个视网膜包括突出内层视网膜层示出，这些基因是由感光体确实表达。对于Gnat2相比，rho和凹陷的相对表达表明，它是由包含在分离的感光层内锥体表达。 recoverin的（RCVRN）在感光层相比，整个视网膜中的表达增加（ 图3B）。 在感光层也用于监测RHO和GNAT2使用免疫印迹按照用抗体供应商提供的方法中的表达，并比较于内视网膜层（ 图4）。分别在整个视网膜并在RD1小鼠在出生后第35天的视网膜注意到缺少RHO和GNAT2 28，杆和视锥细胞的标志物。 <img alt="图1" src="/files/ftp_upload/51954/51954fig1highres.jpg" /> 网络连接古尔1.鼠标视网膜的vibratome切片的示意图。与刀片的vibratome视网膜内层的（A）安装平置式视网膜朝下明胶片上的感光。（B）段，外视网膜（C）条加入明胶。（四）控制感光体的在视网膜的片段的感光体层的分离后的边缘的存在。用于绿色荧光标尺条用于白光显微镜。光感受器位于图像的边缘。（E）的培养感光细胞（后5天）。（F）的高倍面板E的<img alt="图2" src="/files/ftp_upload/51954/51954fig2highres.jpg" width="550" /> RHO和SAG在小鼠感光文化图2.差异表达。核（A）染色（蓝），（B）的 SAG染色（绿色），（C）的 RHO染色（红色）。（D）的合并图像。由于SAG的染色早于RHO染色表示，只有两种细胞双标SAG和RHO（见*）。比例尺：23微米<a href="https://www.jove.com/files/ftp_upload/51954/51954fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51954/51954fig3highres.jpg" /> 图的三个基因在脑和整个视网膜和年龄35天野生型动物的光感受器的表达3.比较。 （A）视紫红质（RHO），S-arrestin的（SAG）和锥转导（Gnat2）。（B）钙结合蛋白（Calb1）和Recoverin（RCVRN）。数据显示为相对表达。相对表达式是一个表达式的值normaliszati​​on该软件强大的多阵列平均（RMA）提供知识数据库KBaSS（http://kbass.institut-vision.org/KBaSS/）后，从芯片数据中获得。 <img alt="图4" src="/files/ftp_upload/51954/51954fig4highres.jpg" width="400" /> 在外层视网膜从一个野生型视网膜在产后35天中的内视网膜和RD1小鼠在出生后第35天ACTB，β-视网膜缺失其表达的图4. RHO和GNAT2的表达肌动蛋白的。 <a href="https://www.jove.com/files/ftp_upload/51954/51954fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <!-- Repeat of the materials table
<table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 材料/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td>评论/说明 </td></tr><tr><td>胞苷5&#39;-diphosphocholin * </td><td> Sigma-Aldrich公司</td><td> C0256 </td><td> 4.7μM胞苷5&#39;-diphosphocholin </td></tr><tr><td>胞苷5&#39;-diphosphoethanolamine * </td><td> Sigma-Aldrich公司</td><td> C0456 </td><td> 2.7μM胞苷5&#39;-diphosphoethanolamine </td></tr><tr><td>亚油酸/牛血清白蛋白（BSA）* </td><td> Sigma-Aldrich公司</td><td> L8384 </td><td> 100微克/毫升的亚油酸/牛血清白蛋白（BSA）的</td></tr><tr><td>三碘左旋甲腺氨酸* </td><td> Sigma-Aldrich公司</td><td> T6397 </td><td> 0.03μM三碘左旋甲腺氨酸</td></tr><tr><td> 96井板</td><td>格雷纳生物一</td><td> 655-095 </td><td></td></tr><tr><td>双目显微镜</td><td>徕卡</td><td> MZ-75 </td><td></td></tr><tr><td> CO 2个独立的（CO 2 -我） </td><td>生活中特chnologies </td><td> 18045054 </td><td></td></tr><tr><td> DMEM </td><td> Life Technologies公司</td><td> 41966029 </td><td></td></tr><tr><td>钳N°5杜蒙</td><td>仿生</td><td> 11254-20 </td><td></td></tr><tr><td>从porcin皮肤类型明胶</td><td> Sigma-Aldrich公司</td><td> G2500 </td><td></td></tr><tr><td>基因芯片</td><td> Affymetrix公司</td><td> U74v2 </td><td></td></tr><tr><td>庆大霉素解决方案</td><td> Life Technologies公司</td><td> 15710049 </td><td></td></tr><tr><td>氢化可的松* </td><td> Sigma-Aldrich公司</td><td> H0888 </td><td> 0.55μM氢化可的松</td></tr><tr><td>胰岛素*（I）的</td><td> Sigma-Aldrich公司</td><td> I1884（ITS）的</td><td> 0.86μM胰岛素（I）的</td></tr><tr><td>木瓜蛋白酶</td><td>沃辛顿，生化</td><td> WOLS03124 </td><td></td></tr><tr><td>聚D-赖氨酸</td><td> Sigma-Aldrich公司</td><td>P-6407 </td><td></td></tr><tr><td>孕酮* </td><td> Sigma-Aldrich公司</td><td> P7556 </td><td> 2.0微米的孕激素</td></tr><tr><td>前列腺素* </td><td> Sigma-Aldrich公司</td><td> P5172 </td><td> 0.28μM前列腺素</td></tr><tr><td>腐胺* </td><td> Sigma-Aldrich公司</td><td> P5780 </td><td> 182μM腐</td></tr><tr><td>手术刀维奇</td><td> EMS </td><td> 72000 </td><td></td></tr><tr><td>剪刀脚</td><td>莫里亚</td><td> 15396-00 </td><td></td></tr><tr><td>丙酮酸钠* </td><td> Sigma-Aldrich公司</td><td> S8636 </td><td> 1mM丙酮酸钠</td></tr><tr><td>亚硒酸钠*（S） </td><td> Sigma-Aldrich公司</td><td> I1884（ITS）的</td><td> 0.29微米的Na 2的SeO 3（S） </td></tr><tr><td>牛磺酸* </td><td> Sigma-Aldrich公司</td><td> T8691 </td><td> 3 mM的牛磺酸</td></tr><tr><td&gt;转*（T） </td><td> Sigma-Aldrich公司</td><td> I1884（ITS）的</td><td> 0.07微米转铁蛋白（T）的</td></tr><tr><td>的vibratome设备</td><td>徕卡</td><td> VT1000-S </td><td></td></tr><tr><td> *补充</td><td></td><td></td><td></td></tr></tbody></table> 表1：特殊材料和设备的使用。 - &gt;

Watch this Scientific Journal Video about Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures at JoVE.com

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

对8岁天小鼠神经视网膜是在4％阿胶块的顶部。感光体层（200微米）通过的vibratome分离后，将感光体后的机械和酶解为培养接种。可用于分子，生化分析或移植的感光层。

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

vibratome-sectioning-mouse-retina-to-prepare-photoreceptor-cultures

Research

JoVE Journal

Neuroscience

16.6K 次观看.  UMR_S 968, Institut de la Vision. 对8岁天小鼠神经视网膜是在4％阿胶块的顶部。感光体层（200微米）通过的vibratome分离后，将感光体后的机械和酶解为培养接种。可用于分子，生化分析或移植的感光层。 

视频: Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

体内 小鼠视网膜血管损伤读数以促进可重复性

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...