Name: two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis
Uploaded: 2021-12-07T13:00:00
Description: Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

这项工作得到了NIH Grant EY12155，EY027193和EY027387对JC的支持。我们感谢Spyridon Michalakis博士（美国帕萨迪纳加州理工学院）和Natalie Chen博士（美国洛杉矶南加州大学）校对手稿。我们还要感谢Seth Ruffins博士（南加州大学，洛杉矶，美国）和Janos Peti-Peterdi博士（南加州大学，洛杉矶，美国）提供必要的设备来收集作者提供的镜头。材料来自：Kasey Rose等人，分离小鼠视网膜中的感光细胞区室进行蛋白质分析，分子神经变性，发表[2017]，[施普林格自然]。

所有实验均根据南加州大学（USC）研究动物护理委员会的当地机构指南进行。
1. 活细胞视网膜剥离法
<ol><li>准备艾姆斯的缓冲液、去皮纸和解剖皿<ol>
<li>使用钝尖虹膜剪刀（或等效的剪刀型），将纤维素滤纸（413级）切割成长方形，尺寸约为5 mm x 2.5 mm。储存插条以备将来使用。</li>
		<li>通过混合一瓶Ames'培养基，2.38克HEPES和0.877克NaCl来制备1升Ames的HEPES缓冲液。...

<ol>
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许多视网膜疾病会影响视杆细胞，导致视杆细胞死亡，并最终导致完全视力丧失37。多年来，人类视网膜变性的遗传和机制起源的很大一部分已经在许多小鼠模型中成功概括。在这种情况下，能够轻松和选择性地将单个杆亚细胞区室与小小鼠视网膜分开，这将大大增强我们对视网膜疾病的局部生化和分子基础的理解。此外，神经视网膜的分层性质，结合杆光感受器独特的高度极?...

棒状光感受器细胞紧密地聚集在神经视网膜的最外层，是昏暗光视觉的一个组成部分。为了充当忠实的光子计数器，杆利用基于G蛋白的信号通路（称为光转导）来产生对单光子捕获的快速，放大和可重复的响应。这种对光的响应最终会触发质膜电流的变化，并随后向视觉系统的其余部分发出信号1。顾名思义，每个杆细胞都具有独特的杆状形状，并表现出高度极化的细胞形态，由外段（OS），内段（IS），细胞体（CB）和突触末端（ST）组成。每个亚细胞区室具有特定的蛋白质机制（膜结合和可溶性）、生物分子特征和蛋白质复合物，这些蛋白质复合物起着至关重要的作用，如视觉光转导、一般内务管理和蛋白质合成以及突触传递2，3。
30多年前，首次观察到亚细胞蛋白的光依赖性相互运动，特别是转导素（远离OS）和停滞素（朝向OS），4，5，6，7。早期，这种观察到的现象受到怀疑，部分原因是免疫组织化学对表位掩蔽的脆弱性8。在2000年代初，通过使用严格而艰苦的物理切片技术证实了刺激依赖性蛋白质易位9。对冷冻的平装啮齿动物视网膜进行连续切向切片，然后进行免疫印迹，结果显示，转导素9，10，arrestin11，12和recoverin13都响应于光的亚细胞再分布。据信，这些关键信号蛋白的光驱动易位不仅调节光转导级联的灵敏度9，14，15，而且还可能对光损伤具有神经保护作用16，17，18。由于视杆细胞中的光驱动蛋白转运似乎对视杆细胞生物学和生理学非常重要，因此允许分离不同亚细胞区室以确定蛋白质分布的技术是有价值的研究工具。
目前，有几种旨在分离杆亚细胞区室的方法。然而，这些方法可能很长且难以复制，或者需要相当数量的视网膜分离物。例如，通过密度梯度离心19制备棒外段（ROS）通常用于将ROS与视网膜匀浆分离。这种方法广泛用于蛋白质印迹，但该过程非常耗时，并且至少需要8-12个小鼠视网膜20。另一方面，冷冻小鼠和大鼠视网膜的连续切向切片已成功用于分离OS，IS，CB和ST9，11，13。然而，这种方法在技术上具有挑战性，因为在切向切片之前，必须完全压平小而高度弯曲的小鼠视网膜以对齐视网膜层。由于有大量的小鼠模型和转基因小鼠概括视觉系统的疾病，因此创建一种可靠，快速且容易地分离单个杆室的技术有望揭示每个专用隔室中发生的生理过程以及健康和疾病中视觉过程背后的机制。
为了促进这些研究，我们描述了两种剥离方法，它们比目前的方案更容易分离杆亚细胞区室。第一种剥离方法改编自一种暴露荧光标记的双极细胞以进行膜片钳记录的技术21，它采用纤维素滤纸从活的，分离的小鼠视网膜中依次去除ROS（图1）。第二种方法改编自从chick22和frog23视网膜中分离出三个主要视网膜细胞层的程序，利用胶带从冻干视网膜中去除ROS和杆内段（RIS）（图2）。这两个程序都可以在1小时内完成，并且非常人性化。我们通过利用来自C57BL / 6J小鼠的黑暗适应和光暴露的视网膜来证明光诱导的杆转导素（GNAT1）和逮捕素（ARR1）的易位，从而验证了这两种分离方案对蛋白质印迹的有效性。此外，使用胶带剥离方法，我们提供了额外的证据，证明我们的技术可用于检查和解决通过免疫细胞化学（ICC）和蛋白质印迹获得的蛋白质定位数据之间的不一致。具体而言，我们的技术表明：1）蛋白激酶C-α（PKCα）亚型不仅存在于双极细胞中，还存在于小鼠ROS和RIS中，尽管浓度低24，25和2）视紫红质激酶（GRK1）主要存在于分离的OS样品中。这些数据证明了我们的两种剥离技术在分离和定量特定视网膜蛋白和视网膜蛋白方面的有效性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>100 mL laboratory or media bottle equipped with a tubing cap adapter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>100% O2 tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>1000mL Bottle Top Filter, PES Filter Material, 0.22 &#956;m</td>
			<td>Genesee Scientific</td>
			<td>25-235</td>
			<td></td>
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		<tr>
			<td>4X SDS Sample Buffer</td>
			<td>Millipore Sigma</td>
			<td>70607-3</td>
			<td></td>
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		<tr>
			<td>50 mL Falcon tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
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		<tr>
			<td>95% O2 and 5% CO2 tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
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		<tr>
			<td>Ames&#8217; Medium with L-glutamine, without bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 (99%, dihydrate)</td>
			<td>Sigma</td>
			<td>C-3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite (Anhydrous calcium sulfate, &#62;98% CaSO4, &#62;2% CoCl2)</td>
			<td>WA Hammond Drierite Co LTD</td>
			<td>21005</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Petri Dish (polystyrene, 35 x 10 mm)</td>
			<td>Falcon-Corning</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Tissue Culture Dish (60 x 15 mm)</td>
			<td>Falcon-Corning</td>
			<td>08-772F</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Scalpel (No. 10, 40 mm)</td>
			<td>VWR</td>
			<td>100499-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Scalpel (No. 11, 40 mm)</td>
			<td>VWT</td>
			<td>100499-580</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl (99%)</td>
			<td>Sigma</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes pellet pestle</td>
			<td>Sigma</td>
			<td>z359971</td>
			<td></td>
		</tr>
		<tr>
			<td>Labconco Fast-Freeze Flasks</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LN2 (liquid nitrogen) + Dewar flask or similar vacuum flask</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M-9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q/de-ionized water</td>
			<td>EMD Millipore</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 (powder)</td>
			<td>J.T. Baker</td>
			<td>4062-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (crystal)</td>
			<td>EMD Millipore</td>
			<td>Sx0420-3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Amresco</td>
			<td>0865</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur EDTA</td>
			<td>EMD</td>
			<td>4005</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur HEPES, Free Acid</td>
			<td>EMD</td>
			<td>5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Reynolds Wrap Aluminum Foil</td>
			<td>Reynolds Brands</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch Magic Tape (12.7 mm x 32.9 m)</td>
			<td>Scotch-3M</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D67501</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrafuge mini centrifuge</td>
			<td>Labnet International, Inc</td>
			<td>C1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue incubation chamber (purchased or custom made)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>J.T.Baker</td>
			<td>4103-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Signma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>VirTis Benchtop 2K Lyophilizer or equivalent machine</td>
			<td>SP Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Grade 413 Filer Paper (diameter 5.5 cm, pore size 5 &#956;m)</td>
			<td>VWR</td>
			<td>28310-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Grade 1 Qualitative Filter Paper (diameter 9 cm, pore size 11 &#956;m)</td>
			<td>Whatman/GE Healthcare</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide bore transfer pipet, Global Scientific</td>
			<td>VWR</td>
			<td>76285-362</td>
			<td></td>
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</table>

two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis

视杆光感受器是具有不同区室的高度极化感觉神经元。小鼠杆长（~80μm）和薄（~2μm），横向堆积在视网膜的最外层，即光感受器层，导致类似的亚细胞区室对齐。传统上，冷冻的平板视网膜的切向切片已被用于研究不同杆室内蛋白质的运动和定位。然而，杆状小鼠视网膜的高曲率使得切向切片具有挑战性。在对区室间蛋白质转运研究的推动下，我们开发了两种剥离方法，可以可靠地分离出杆外段（ROS）和其他亚细胞区室用于蛋白质印迹。我们相对快速和简单的技术提供富集和亚细胞特异性级分，以定量测量正常视杆中重要光感受器蛋白的分布和再分布。此外，这些分离技术也可以很容易地用于分离和定量研究健康和退化视网膜中其他细胞层的蛋白质组成。

本策略的开发是为了提供相对快速和简单的方法来分离和分析特定杆亚细胞区室中的蛋白质，以进行蛋白质印迹分析。我们应用了两种顺序剥离技术（图1 和 图2），然后进行免疫印迹，以证明这些方法可以可靠地用于检测黑暗和光适应动物中杆转导素（GNAT1）和停滞素（ARR1）的已知分布。为了验证我们的两种方案在精确分离杆亚细胞区室而不污染其他...

Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

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

该协议提出了两种技术来分离小鼠杆光感受器的亚细胞区室以进行蛋白质分析。第一种方法利用活视网膜和纤维素滤纸来分离杆外段，而第二种方法采用冻干视网膜和胶带剥离杆内段和外段层。

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 ...

这里展示的两种剥离技术可分离单个杆亚细胞区室，并可以帮助揭示健康和患病杆细胞中每个特殊区室中发生的重要生理过程。从小鼠视网膜中分离出不同的视杆细胞区室可能具有挑战性。这些简单的技术使用廉价且司空见惯的实验室材料来可靠地分离杆亚细胞区室以进行蛋白质分析。首先，将一张滤纸放入装满100%氧气气泡的Ames HEPES缓冲液的培养皿中。然后，使用宽孔转移移液器，从两到三个月大的C57 Black 6J小鼠中移出一个解剖出的视网膜矩形。将分区的视网膜向上凹陷，并确保光感受器朝下朝向培养皿的底部。然后，使用镊子轻轻抓住半视网膜的侧面，并将其移动到滤纸的顶部。一旦视网膜居中，向上移动滤纸，使切成两半的视网膜接触滤纸以产生杆外段，或ROS以产生滤纸粘附。小心地将带有视网膜的滤纸从Ames HEPES缓冲液中提起。将滤纸的底部放在纸巾上，轻拍两到三次以干燥。将一滴Ames HEPES加入视网膜滤纸的侧面，然后将滤纸再次放在纸巾上晾干。接下来，将带有视网膜的滤纸放回培养皿中，然后用镊子轻轻地将切成两半的视网膜的所有边缘向上推，使其远离所有侧面的滤纸。小心地将视网膜从滤纸上剥离，仅接触视网膜的极端周边，以保持其结构完整性。然后，从培养皿中取出滤纸，除去纸巾上多余的液体，确保与视网膜接触的一侧不会接触纸巾。将带有部分ROS层的滤纸放入冰上的标记管中，并保持剥离的视网膜浸没在Ames HEPES缓冲液中。对这个减半的视网膜重复剥离过程大约七到八次，以去除整个ROS层。每次剥离后，将滤纸放入同一管中，以组合从一个对半视网膜分离出来的所有ROS。将装有所有分离ROS滤纸剥离的管子放在冰上，以便立即使用。使用镊子将剩余的剥离的视网膜转移到适当标记的管中，并将管子放在冰上以立即使用。要准备用于冻干的视网膜样品，请放置一张滤纸并将减半的视网膜转移到装有冷Ringer缓冲液的培养皿中。接下来，使用镊子小心地翻转视网膜，使其朝上凹陷，然后将其移动到滤纸的顶部。将滤纸的边缘向上抬起，使视网膜的一半接触到滤纸。继续将滤纸从林格的溶液中取出。然后，将滤纸的底部放在纸巾上，小心地轻拍两到三次以除去多余的液体。接下来，用镊子拿起滤纸，在带有视网膜的滤纸侧面加入一滴冷铃声。同样，将滤纸放在纸巾上以除去多余的液体。将每个粘附的视网膜和滤纸样品存放在装满冷铃声的培养皿中，直到准备好冷冻所有样品。然后，使用镊子将每个样品从冷的Ringer缓冲液中取出，确保镊子仅接触滤纸边缘，避免视网膜减半。将每张滤纸的底部放在纸巾上，以除去多余的液体。然后，将一滴冷PBS添加到滤纸的侧面，其中有视网膜粘附，并再次将滤纸轻拍在纸巾上。将所有滤纸放入培养皿中，并使用不起毛的薄纸吸走滤纸周围多余的液体。然后，用铝箔紧紧包裹整个培养皿，并确保铝箔的边缘固定并平稳地压入培养皿的底部。在铝箔盖上刺穿一些0.1至0.2毫米的孔。接下来，使用金属钳，逐渐将铝箔覆盖的培养皿降低到液氮中。将样品保存在液氮中，直到准备好冻干。对于冻干，将铝箔覆盖的培养皿放入冷冻干燥瓶中，并按照制造商的协议将其连接到冻干机上。冻干视网膜30分钟。冻干后，通过小心地在冻干视网膜顶部放置一小块胶带并用镊子施加轻微压力以确保其与感光器层结合来收集杆外段和内段。慢慢剥离磁带，确保 ROS 和 RIS 都粘附在磁带上。断裂表面将出现一层薄薄的白色薄膜。这是RIS。要分离此RIS，请放置另一块胶带，然后将胶带向下推到断裂的表面上。然后，将只有橙粉色层的胶带放入标有ROS的微量离心管中，并将带有薄薄白色层的胶带放入另一根标有RIS的管中。接下来，使用胶带，通过剥离厚厚的白色层来收集位于滤纸上的剩余视网膜组织。然后，将此隔离物放入标有OIS的管中。如果当天使用，请在室温下储存冻干视网膜中的分离层。在适应黑暗的动物中，GNAT1和ARR1的分布与它们已知的暗态分布非常匹配，其中GNAT1信号在ROS隔离中明显最强，而ARR1信号在ROS隔离中更强。对GNAT1和ARR1信号的定量显示，ROS和ROS样品中GNAT1和ARR1的暗光条件之间存在显着差异。在黑暗和光适应的样品中，G beat 5S，细胞色素C和肌动蛋白信号从ROS样品中排除，表明没有来自其他细胞层的污染。此外，G beta 5L信号在ROS样品中清晰可见。对完整和剥离的冻干视网膜表面的扫描电镜检查表明，在用胶带剥离之前，完整冻干视网膜的表面与特征圆柱形ROS紧密匹配。在最初的胶带剥离后，ROS和RIS似乎被完全去除，正如残留的，剥离的冻干视网膜表面上存在的均匀核层所证明的那样。随后的胶带剥离从剥离层的自由表面上除去RIS。该技术还表明，GRK1免疫反应性在光暴露的视网膜中的ROS样品中最强烈，而在OIS样品中不存在。相反，ROS和RIS样品显示出PKCα的微弱信号，这在ROS或RIS中通常不能通过免疫荧光染色检测到。最后，次优的胶带剥离可能会产生轻微污染的样品。RIS样品被一些OIS样品污染，产生G beta 5L和G beta 5S波段，以及更高的PKC alpha信号。冻干视网膜时必须小心。如果使用不正确的冻干技术，视网膜可能会融化，从而无法通过胶带分离不同的杆亚细胞层。将这种胶带剥离方法与qRT-PCR结合使用，可以将光感受器转录本与视网膜的其余部分分离。

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

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 ...

科研

神经科学

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

Methods for the Modulation and Analysis of NF-ÃƒÅ½Ã‚ÂºB-dependent Adult Neurogenesis

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

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). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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 ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

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

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

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 ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

你在哪里切的问题: 眼地标性小鼠视网膜空间定位的解剖与分析指南

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...