Name: evisceration of mouse vitreous and retina for proteomic analyses
Uploaded: 2011-04-03T15:00:00
Duration: 2 min 39 s
Description: Watch this Scientific Journal Video about Evisceration of Mouse Vitreous and Retina for Proteomic Analyses at JoVE.com

争取视线和研究防盲提供了资金。

1。眼前段解剖。 <ol><li>角膜缘后巩膜组织掌握0.22 forcep和全球（眼球）是稳定的。 </li><li>一个显微刀片是用来在从角膜缘角膜缘的角膜线性切口。 </li><li>然后用0.12科利柏forcep把握角膜切口。 </li><li>前房液，然后吸收与Weck - CEL手术矛。 </li></ol> 2。镜头剜。 <ol><li> 0.12科利柏forcep抓角膜用于稳定全球。 </li><li>一个良好的弯针持有人（或弯曲敷料钳）插入后面的镜头走向全球的后路方面。持针器是局部封闭，并向前拉。巩膜，推动通过角膜切口的镜头而离开眼球壁的完整性的外部表面上使用镊子施加压力。玻璃体出现一个半透明的凝胶，部分附着在镜头上。 </li><li>镜头玻璃体组织，然后放入含有20微升PBS溶解蛋白酶抑制剂的鸡尾酒（罗氏）一个过滤离心管。 </li></ol> 3。视网膜剜。 <ol><li>继续稳定与0.12科利柏抓角膜切口钳全球。 </li><li>一个良好的弯针持有人（或弯曲敷料钳）是放在全球尽可能远后，附近的视神经。持针器是局部封闭，并向前拉。巩膜，视网膜推动通过角膜切口的外部表面上使用镊子施加压力。作为一个半透明的凝胶，附着于视网膜玻璃体出现。视网膜是为黄色，吻合血管的组织的可视化。色素组织的任何条可用镊子剥离。 </li><li>玻璃体视网膜组织，然后放入过滤离心机包含镜头玻璃体组织管。 </li></ol> 4。过滤的离心。 <ol><li>过滤离心管放置在台式离心机。 14000 X克分拆为12分钟。 </li><li>从下腔，这是玻璃体吸洗脱。视网膜仍然在上议院。这些样本可以用于蛋白质研究。 </li></ol> 5。代表性的成果经SDS - PAGE（图2）玻璃体样本进行了分析。样品也被用于酶活性测定（图3）。 <img src="/files/ftp_upload/2795/2795fig1.jpg" alt="图1"/> 图1。老鼠眼图。横断面示意图显示了人类和小鼠的眼睛中由于其较大的镜头鼠标眼睛玻璃体体积相对较小。 <img src="/files/ftp_upload/2795/2795fig2.jpg" alt="图2"/> 图2一维的SDS - PAGE鼠标玻璃体和视网膜。鼠标玻璃体蛋白质图谱，13.6毫克/毫升（1巷），和视网膜，11.35毫克/毫升（2巷）。凝胶电泳在150千伏为45分钟，使用Flamingo荧光凝胶染色的染色和可视化使用VersaDoc成像系统（BioRad公司，大力士，CA）。 <img src="/files/ftp_upload/2795/2795fig3.jpg" alt="图3"/> 图3。超氧化物歧化酶（SOD）酶的活性在小鼠和人的玻璃体 。剜DBA和白化小鼠收集的玻璃体和视网膜上执行测量总SOD活性（SOD活性比色法aasay套件，＃AB65354，Abcam公司，公司，剑桥，马萨诸塞）。这是相比，在人类玻璃体核心和人类从验尸报告的人类捐助眼睛的视网膜收集到的确定的相对活性SOD活性。手动吸气，用一个23号针头，11日和开花的眼睛和切割组织，远离复杂的RPE /脉络膜视网膜收集，未经稀释的玻璃体核心样品。虽然检测不区分超氧化物歧化酶异构体，玻璃体SOD活性很可能反映SOD3，因为它是唯一的外亚型 12视网膜SOD活性很可能反映了所有三个超氧化物歧化酶亚型：是细胞内的SOD1，线粒体SOD2 ，外12 SOD3。高度敏感的酶检测，包括这种检测SOD，蛋白质的交叉污染的可能性是可能的，需要进一步研究。误差棒代表教统局局长。

<ol>
	<li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel.">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog. Retin. Eye Res. 19, 323-344 (2000).</li><li>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P. &. a. m. p. ;. a. m. p., Feener, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+vitreous+proteome+in+diabetes+without+diabetic+retinopathy+and+diabetes+with+proliferative+diabetic+retinopathy.">Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.</a> J. Proteome Res. 7, 2516-2525 (2008).</li><li>Izuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+SOD+and+VEGF+are+increased+in+vitreous+bodies+from+proliferative+diabetic+retinopathy+patients.">Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients.</a> Mol. Vis. 15, 2663-2672 (2009).</li><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pharmacologic+vitreolysis.">Molecular biology of pharmacologic vitreolysis.</a> Trans. Am. Ophthalmol. Soc. 103, 473-494 (2005).</li><li>Smith, R. S., Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene. Ther. Mol. Biol. 11B, 229-262 (2007).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Human+Vitreous+Body+Elements+for+Proteomic+Analysis.">Dissection of Human Vitreous Body Elements for Proteomic Analysis.</a> J Vis Exp. , (2011).</li><li>Ciechanowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+synthesis+is+not+the+reason+for+decreased+activity+of+extracellular+superoxide+dismutase+in+patients+with+diabetes.">Impaired synthesis is not the reason for decreased activity of extracellular superoxide dismutase in patients with diabetes.</a> Arch Med Res. 36, 148-153 (2005).</li></ol>

按照协会的研究和视觉眼科眼科及视觉研究中使用动物的声明进行了动物实验。

然而，转基因小鼠视网膜和玻璃体视网膜疾病调查的重要模式。6-10鼠标玻璃体，包括比较由于在小鼠眼大镜头玻璃体人类眼睛的比例明显较小。5这使得很难分离和纯化鼠标玻璃体。了解蛋白质与疾病，如增生性糖尿病视网膜病变，年龄相关性黄斑变性，葡萄膜炎，视网膜脱离玻璃体变化会给机制的洞察力，他们的进步。这种可视化的实验协议，提供了一种手段获取和净化鼠标玻璃体，同时最大限度的酶和蛋白组学分析的蛋白质产量。

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>产品编号</th><th>评论</th></tr><tr><td> 15 °，BD海狸显微刀片</td><td>流式细胞 - 迪金森</td><td> 374881 </td><td></td></tr><tr><td> PBS，pH值7.4 </td><td> Invitrogen公司</td><td> 70011-044 </td><td></td></tr><tr><td> 0.22 Castroviejo精细缝合钳</td><td> STORZ Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0.12科利柏钳</td><td> STORZ Ophthalmics </td><td> 2 / 132 </td><td></td></tr><tr><td>单片机离心过滤Ultracel YM - 100或Y​​M - 50 </td><td> Millipore公司</td><td> 42412，42415 </td><td></td></tr><tr><td> SOD活性比色法检测试剂盒</td><td> Abcam公司</td><td> Ab65354 </td><td></td></tr><tr><td> Weck - CEL手术矛</td><td>美敦力公司</td><td> 0008680 </td><td></td></tr><tr><td>蛋白酶抑制剂的鸡尾酒</td><td>罗氏公司</td><td> 11 836 170 001 </td><td></td></tr><tr><td>火烈鸟荧光凝胶染色</td><td> Bio - Rad公司</td><td> 161-04910 </td><td></td></tr></tbody></table>

evisceration of mouse vitreous and retina for proteomic analyses

虽然作为一个重要的遗传性视网膜疾病的遗传模型小鼠视网膜出现，鼠标玻璃体仍然有待探索。玻璃体是一个高水覆人工以及眼蛋白质在疾病过程中积累的视网膜细胞外基质 。1-3异常玻璃体和视网膜之间的相互作用的几种疾病，如视网膜脱离，增殖性糖尿病视网膜病变，葡萄膜炎，和增生性玻璃体视网膜病变的基础。 4相对鼠标玻璃体体积明显比人类的玻璃体（图1），是因为鼠标镜头中占有近75％的眼睛。5这使得鼠标玻璃体生化研究具有挑战性。在这个视频文章中，我们目前的技术来剖析和隔离鼠标玻璃体视网膜，这将允许使用转基因小鼠模型，以更清楚地界定这种细胞外基质的作用，在玻璃体视网膜疾病的发展。

Watch this Scientific Journal Video about Evisceration of Mouse Vitreous and Retina for Proteomic Analyses at JoVE.com

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

剥离技术说明玻璃体，视网膜，并从小鼠眼晶状体，通过离心分离，并与蛋白质分析表征剜。

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Research

JoVE Journal

Biology

Horizontal Slice Preparation of the Retina

视网膜水平切片制备

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

科研

生物学

Electrophoretic Separation of Proteins

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Immunoblot Analysis

免疫印迹分析

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Staining Proteins in Gels

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Digital Microfluidics for Automated Proteomic Processing

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

轨迹数据分析行人时空活性研究

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...