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><tbody><tr><td>Cytidine 5&amp;prime;-diphosphocholin*</td><td>Sigma-Aldrich</td><td>C0256</td><td>4.7 &amp;micro;M Cytidine 5&amp;prime;-diphosphocholin</td></tr><tr><td>Cytidine 5&amp;prime;-diphosphoethanolamine*</td><td>Sigma-Aldrich</td><td>C0456</td><td>2.7 &amp;micro;M Cytidine 5&amp;prime;-diphosphoethanolamine</td></tr><tr><td>Linole&amp;iuml;c acid/ bovine serum albumin (BSA)*</td><td>Sigma-Aldrich</td><td>L8384</td><td>100 &amp;micro;g/ml linole&amp;iuml;c acid/ bovine serum albumin (BSA)</td></tr><tr><td>Triiodo-L-thyronine*</td><td>Sigma-Aldrich</td><td>T6397</td><td>0.03 &amp;micro;M Triiodo-L-thyronine</td></tr><tr><td>96-well plates</td><td>Greiner bio-one</td><td>655-095</td><td></td></tr><tr><td>Binocular microscope</td><td>Leica</td><td>MZ-75</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; independent (CO&lt;sub&gt;2-&lt;/sub&gt;i)</td><td>Life Technologies</td><td>18045054</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>41966029</td><td></td></tr><tr><td>Forceps n&amp;deg;5 Dumont</td><td>Bionic</td><td>11254-20</td><td></td></tr><tr><td>Gelatin from porcin skin type A</td><td>Sigma-Aldrich</td><td>G2500</td><td></td></tr><tr><td>GeneChip&amp;nbsp;</td><td>Affymetrix</td><td>U74v2</td><td></td></tr><tr><td>Gentamicin solution</td><td>Life Technologies</td><td>15710049</td><td></td></tr><tr><td>Hydrocortison*</td><td>Sigma-Aldrich</td><td>H0888</td><td>0.55 &amp;micro;M hydrocortison</td></tr><tr><td>Insulin* (I)</td><td>Sigma-Aldrich</td><td>I1884 (ITS)</td><td>0.86 &amp;micro;M insulin (I)</td></tr><tr><td>Papain</td><td>Worthington-biochem</td><td>WOLS03124</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P-6407</td><td></td></tr><tr><td>Progesterone*</td><td>Sigma-Aldrich</td><td>P7556&amp;nbsp;</td><td>2.0 &amp;micro;M progesterone</td></tr><tr><td>Prostaglandin*</td><td>Sigma-Aldrich</td><td>P5172</td><td>0.28 &amp;micro;M prostaglandin</td></tr><tr><td>Putrescine*</td><td>Sigma-Aldrich</td><td>P5780</td><td>182 &amp;micro;M putrescine</td></tr><tr><td>Scalpel Albion</td><td>EMS</td><td>72000</td><td></td></tr><tr><td>Scissor</td><td>Moria</td><td>15396-00</td><td></td></tr><tr><td>Sodium pyruvate*</td><td>Sigma-Aldrich</td><td>S8636</td><td>1 mM sodium pyruvate</td></tr><tr><td>Sodium selenite* (S)</td><td>Sigma-Aldrich</td><td>I1884 (ITS)</td><td>0.29 &amp;micro;M Na&lt;sub&gt;2&lt;/sub&gt;SeO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;&amp;nbsp; (S)</td></tr><tr><td>Taurine*</td><td>Sigma-Aldrich</td><td>T8691</td><td>3 mM taurine</td></tr><tr><td>Transferrin* (T)</td><td>Sigma-Aldrich</td><td>I1884 (ITS)</td><td>0.07 &amp;micro;M transferrin (T)</td></tr><tr><td>Vibratome apparatus</td><td>Leica</td><td>VT1000-S</td><td></td></tr><tr><td colspan="4">* Supplements</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.5K 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

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

器官型培养的全厚成年猪视网膜

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

科研

神经科学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

生物学

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 ...

研究小鼠分裂视网膜中的双极和水平神经元细胞

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

作者聚焦：使用 Split Retina 技术增强访问和加速实验 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

的vibratome切片小鼠视网膜准备感光文化

摘要

探索更多视频

器官型培养的全厚成年猪视网膜

量化的活动顺调控元件外植体电穿孔

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

成像钙

成年小鼠视网膜电生理研究水平切片的制备

视网膜平片准备成像和免疫组化优化方案

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

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂