Name: immunohistochemical and calcium imaging methods in wholemount rat retina
Uploaded: 2014-10-13T13:00:00
Description: Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

我们感谢博士学Stella和海伦VUONG向钙成像协议的贡献。我们感谢K.医生表拍戏面试场景。我们感谢阿琳平野博士，她对稿件的意见。该研发项目是由作者在医学大卫格芬医学院在加州大学洛杉矶分校进行的，是可能由被授予和管理由美国陆军医学研究与装备司令部（USAMRMC）的合同协议，远程医疗与先进技术研究中心（TATRC），在德特里克堡，下合同编号MD：W81XWH-10-2-0077。支持这些研究也来自美国国立卫生研究院EY04067和VA价值评议（NB）。 NCB是弗吉尼亚州职业研究科学家。

所有实验均进行了按照由人类善待实验动物和加州大学洛杉矶分校（UCLA）的动物研究委员会，美国公共卫生服务政策颁布实验动物福利的准则。雄性和雌性的成年Sprague-Dawley大鼠的3-5周龄之间（查尔斯河实验室，威尔明顿，马萨诸塞州）被使用。 1，动物和组织准备Wholemount视网膜和免疫组化协议<ol><li>准备以下的材料和工具：解剖显微镜，镊子2用很细的技巧，剪刀，纤维素...

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在这篇文章中，我们描述了两种不同的方法：1）免疫组化染色显示荧光4定位于神经节细胞的视网膜wholemounts，和2）钙成像分析钙动力学的视网膜神经节细胞及其轴突。 使用wholemounts免疫组已被用于揭示蛋白质的定位在啮齿动物视网膜20-23。不过，也有一些限制。染色的质量在很大程度上依赖于该抗体的识别其相应的抗原和其穿透视网膜组织对感兴趣的特定层的能力的...

视网膜神经节细胞（RGC的）表达L，N，P / Q-和T-型VGCC通过整个细胞的这些成分的药理学阻断测定Ca通道电流1,2。 VGCC是涉及递质释放，基因转录，细胞调节和突触可塑性3,4,5该跨膜多聚体蛋白。官能VGCCs是由至少三种不同类的亚基组成：大，跨膜α1孔形成在建立通道的生物物理和药理性质，主要是细胞外的辅助α2δ亚基和胞内β亚基的亚基。后两种形式的异配合不同的α 亚单位和改变门控动力学的渠道质膜6和贩运。 在最近的几十年中，许多技术已被用于研究蛋白质expre裂变，如免疫组织化学，酶联免疫吸附测定法，western分析和流式细胞术。这些技术需要使用特定的抗体对于感兴趣的给定的蛋白质的检测，并提供本地化和特异性蛋白质的分布在不同组织中的有力工具。用于检测和定量特定蛋白质的mRNA表达水平的技术，例如Northern印迹分析，RT-PCR，实时定量RT-PCR， 原位杂交，cDNA芯片和核糖核酸酶保护测定法提供了另一种方法，当抗体是不容易特定的蛋白质可用，或者表达水平低7。然而，一个限制，以使用这样的分子生物学技术是基因序列的所需鉴定。 在视网膜本地化蛋白，免疫组织化学可wholemount视网膜进行。由于视网膜神经节细胞的可及性，wholemount准备提供给特定蛋白质的本土化研究，以研资局胞体和轴突一个很好的平台。 除了其定位，VGCCs在视网膜神经节细胞的一些功能特性可以通过使用钙成像技术来证明。我们描述了钙成像协议选择性标记视网膜神经节细胞与钙指示剂染料测量细胞内钙离子动态。不同VGCCs来在不同的细胞区室中的钙信号的贡献可以分离与使用亚型特异性钙通道阻滞剂。 也许这里描述的钙成像技术的最有利的方面之一是，从多个视网膜神经节细胞和其轴突同时和独立地记录的能力。虽然许多生理技术，如全细胞膜片钳记录，提供膜电流的高时间分辨率的录音，躯体或采取这些轴突源记录ê电流不能歧视和录音只能从每次的单个神经元的进行。多电极阵列（MEAs）中能够从许多细胞同时记录的尖峰，但既不能检测或识别的激活，例如，不同的钙离子通道的亚型。 MEAS从细胞位于接近该给定的电极8和记录从细胞中产生大的尖峰9优先记录。光学成像方法提供了一种替代的策略，使细胞的整个种群可与从单细胞微电极和膜片钳记录和MEA记录中获得的信息结合起来的同时和独立记录。虽然这里所描述的钙成像技术被用于研究视网膜神经节细胞的钙动力学，膜片钳和多边环境也可使用在平行于进一步阐明离子电流和视网膜神经节细胞的动作电位发放。 <pCLASS =“jove_content”&gt;由于流离失所无长突细胞组成的神经元群在小鼠视网膜10的神经节细胞层的约60％，我们的目标是利用装载技术，选择性地标记与视网膜神经节细胞的合成钙指示剂染料wholemount准备。虽然合成钙指示剂染料为细胞内钙离子动力学研究一个很好的平台，它的广泛使用已经阻碍无法给定的网络中有效地加载神经元的特定人群。如批量加载11和电穿孔8,12许多技术已被执行的加载单元的整个人群中，然而，这种技术不特定的细胞类型之间进行区分。遗传编码的钙指标提供以选择性标记的细胞的特定群体的能力，但是，这样的方法要求的转基因动物13的产生。我们的技术描述了其实​​现方法具d，来选择经由视神经断端注射钙指示剂染料的wholemount准备标记视网膜神经节细胞。 综上所述，本文中介绍的结构和生理技术提供研究视网膜神经节细胞和其轴突的定位和VGCCs的钙信号的作用的平台。

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight scissors</td>
			<td style="width:112px;">WPI</td>
			<td style="width:119px;">14124-G</td>
			<td style="width:229px;">dissecting tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight Forceps</td>
			<td style="width:112px;">WPI</td>
			<td style="width:119px;">501985</td>
			<td>dissecting tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">curved iris scissors</td>
			<td style="width:112px;">WPI</td>
			<td>504487</td>
			<td>dissecting tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellulose filter paper</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:119px;">HABP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hibernate A</td>
			<td>Invitrogen</td>
			<td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=A1247501">A12475-01</a></td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield</td>
			<td>Vector</td>
			<td>H-1000&#160;</td>
			<td><a href="https://www.google.com/url?sa=t&amp;rct=j&amp;q=&amp;esrc=s&amp;source=web&amp;cd=1&amp;cad=rja&amp;sqi=2&amp;ved=0CC0QFjAA&amp;url=http%3A%2F%2Fwww.vectorlabs.com%2Fcatalog.aspx%3FcatID%3D279&amp;ei=VAHbUb_fBceeiQLK2IHIDw&amp;usg=AFQjCNHijFTTB8p19FMPQ5hg_qTawXU9mA&amp;sig2=FJCfB9fzKR_TmjxoGvhLcg&amp;bvm=bv.48705608,d.cGE">Mounting Media for Fluorescence</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 pentapotassium</td>
			<td>Invitrogen</td>
			<td>F-14200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td>Abbott Laboratories</td>
			<td>05260-05</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope slide</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">22-178-277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss LSM 5 Pascal microscope</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axioplan 40x (NA 0.8) objective lens</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:112px;">Molecular Probes</td>
			<td style="width:119px;">A-11034</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RBPMS</td>
			<td style="width:112px;">ProSci, Poway, CA</td>
			<td style="width:119px;"></td>
			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
		</tr>
	</tbody>
</table>

immunohistochemical and calcium imaging methods in wholemount rat retina

在本文中，我们描述了工具，试剂，并且需要的实际步骤：1）成功制备wholemount视网膜的免疫组化和，2）钙成像的电压门控性钙通道（VGCC）在视网膜介导的钙信号研究神经节细胞。钙成像方法，我们描述关于位移的无长突细胞在神经节细胞层的非特异性加载规避问题。

用特异性抗体免疫标记，为研究在视网膜和钙成像技术感兴趣的特定蛋白质的定位平台允许的VGCCs在视网膜神经节细胞和它们的轴突的钙动力学的贡献的研究。 通过用抗体对神经节细胞蛋白RBPMS（RNA具有多个剪接结合蛋白），该选择性标记的RGC 19，我们能够显示，荧光4标记限定为神经节细胞（ 图1）。残端注射不会导致所有的神经节细胞的如可预期的，从...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

免疫组化协议，用于研究在视网膜特定蛋白质的定位。钙成像技术被用来研究视网膜神经节细胞及其轴突钙的动态。

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Research

JoVE Journal

Neuroscience

Functional Calcium Imaging in Developing Cortical Networks

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

科研

神经科学

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Subcellular Imaging of Neuronal Calcium Handling In Vivo

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus (Video) | JoVE

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging (Video) | JoVE

电刺激平装视网膜中的钙成像

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...