Name: quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in munich wistar rats
Uploaded: 2013-04-17T12:00:00
Description: Watch this Scientific Journal Video about Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats at JoVE.com

笔者想感谢博士西尔维娅乙坎波斯Bilderback和乔治J罗德完成手术，放置静脉通路线。他们也想感谢萨拉É断奶维持慕尼黑的Wistar殖民地组成的Simonsen和Frömter的菌株。

1。共轭大鼠血清白蛋白磺酸罗丹明101磺酰氯（德州红） <ol><li>溶解100mg大鼠血清白蛋白（RSA）6.667毫升100毫米碳酸氢钠pH值9.0;终浓度为15毫克/毫升，50毫升锥形管。 </li><li>将溶液在冰/水的烧杯中，并冷却至0至4℃之间</li><li>高品质的无水二甲基甲酰胺（DMF）中加入200微升10毫克小瓶德克萨斯红磺酰氯（TRSC）培养基上，在15秒的涡流。 </li><li>的涡RSA解决方案培养基上，并添加溶解TRSC。 </li><li>余音...

<ol>
	<li>Russo, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normal+kidney+filters+nephritic+levels+of+albumin+retrieved+by+proximal+tubule+cells;+retrieval+is+disrupted+in+nephritic+states.">The normal kidney filters nephritic levels of albumin retrieved by proximal tubule cells; retrieval is disrupted in nephritic states.</a> Kidney International. 71, 504 (2007).</li><li>Russo, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+tubular+uptake+explains+albuminuria+in+early+diabetic+nephropathy.">Impaired tubular uptake explains albuminuria in early diabetic nephropathy.</a> Journal of the American Society of Nephrology. 20 (3), 489 (2009).</li><li>Sandoval, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+factors+influence+glomerular+albumin+permeability+in+rats.">Multiple factors influence glomerular albumin permeability in rats.</a> Journal of the American Society of Nephrology. 23 (3), 447 (2012).</li><li>Tojo, A., Endou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarenal+handling+of+proteins+in+rats+using+fractional+micropuncture+technique.">Intrarenal handling of proteins in rats using fractional micropuncture technique.</a> American Journal of Physiology. 263, 601 (1992).</li><li>Asgeirsson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+sieving+of+three+neutral+polysaccharides,+polyethylene+oxide+and+bikunin+in+rat:+Effects+of+molecular+size+and+conformation.">Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat: Effects of molecular size and conformation.</a> Acta Physiologica. 191 (3), 237 (2007).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+endocytosis+in+vivo+using+intravital+two-photon+microscopy.">Quantifying endocytosis in vivo using intravital two-photon microscopy.</a> Methods in Molecular Biology. 440, 389 (2008).</li><li>Dunn, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+imaging+of+renal+function+by+multi-photon+microscopy.">Live-animal imaging of renal function by multi-photon microscopy.</a> Curr. Protoc. Cytom. Chapter 12, Unit 12.9 (2007).</li></ol>

这里强调的步骤代表我们的感受是，将产生一致和准确的渗透值，因为他们规避以下陷阱： <ol><li>散射：使用红色发光的荧光基团可以更有效地收集光，因为较长的波长的光子是不容易分散。使用绿色或蓝色发光荧光团将在GSC的引入更大的变化，因为增加了毛细血管袢和鲍曼的空间变异强度值。 </li><li>图像深度：尽管深通常是收集3D数据集（通常是总深度在30-45微米之间）;高10-15微米代表震源?...

<table border="1">
 <tbody>
 <tr>
 <td>Olympus Floview 1000 confocal/Multi-photon microscope</td>
 <td>Olympus America</td>
 <td></td>
 <td>Filters for detectors: Blue 430/100, Green 525/50, Red 605/90</td>
 </tr>
 <tr>
 <td>Mode-Locked Ti:Sapphire Mai Tai Laser</td>
 <td>Spectra-Physics</td>
 <td></td>
 <td>Tunable excitation wavelengths: ~750-1150 nm</td>
 </tr>
 <tr>
 <td>Gallium arsenide phosphide photodetectors</td>
 <td>Hamamatsu Corp</td>
 <td></td>
 <td>Note: Front or Side mounted configurations available.</td>
 </tr>
 <tr>
 <td>Metamorph Image processing Software</td>
 <td>Molecular Dynamics</td>
 <td></td>
 <td>Note: Version 6.1r1</td>
 </tr>
 <tr>
 <td>Microsoft Excel</td>
 <td>Microsoft Corportation</td>
 <td></td>
 <td>2007 version</td>
 </tr>
 <tr>
 <td>Handling Forceps</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78266-04</td>
 <td></td>
 </tr>
 <tr>
 <td>Mayo Dissecting Scissors</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72962</td>
 <td></td>
 </tr>
 <tr>
 <td>CA Micro scissors Model 1C300</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78180-1C3</td>
 <td></td>
 </tr>
 <tr>
 <td>Kelly Hemostatic Forceps (straight)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72930</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Jacket Blanket + Heating Pad</td>
 <td>Gaymar</td>
 <td></td>
 <td>T/Pump PN 11184-000 Blanket-66N111CC</td>
 </tr>
 <tr>
 <td>Repti-Therm Under Tank Heater</td>
 <td>ZooMed</td>
 <td></td>
 <td>RH-4</td>
 </tr>
 <tr>
 <td>Texas Red Sulfonyl Chloride</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>Cat# T-353</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat Serum Albumin</td>
 <td>Sigma Aldrich</td>
 <td>Cat# A-6272</td>
 <td></td>
 </tr>
 <tr>
 <td>High Quality Anhydrous DMF</td>
 <td>Sigma Aldrich</td>
 <td>Cat# 270547</td>
 <td></td>
 </tr>
 <tr>
 <td>Strate-Line Autoclave Tape</td>
 <td>Fisher Scientific</td>
 <td>Cat# 11-889-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Willco-dish Coverslip Bottom Dishes (50 mm/40 mm coverslip)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 70665-07</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in munich wistar rats

肾疾病，尿中丢失大必不可少的大分子，如血清白蛋白，一直被认为由足细胞的通透性屏障，血管内皮细胞，和以协调方式工作的基底膜的改变引起的。数据从我们的实验室中使用活体双光子显微镜揭示一个更具渗透性的肾小球滤过屏障（GFB），比以前认为的生理条件下，过滤白蛋白发生在早期的子细胞，称为肾小管上皮细胞（PTC）1,2检索， 3。 用以前的技 ​​术研究肾小球滤过和建立滤过屏障特性，包括微注射，这些早期的管状部分的内腔中的流体含量的采样和分析4。这些研究确定管腔液中的白蛋白浓度几乎不存在相应密切通常是在尿中检测到。然而，葡聚糖聚合物，用这种技术定义的大小的表征显示大小类似血清白蛋白的管状腔体和尿中有更高的水平;提示通透性增加5。 本文是一个详细的轮廓，所使用的技术，直接可视化和量化肾小球荧光体内白蛋白通透性 。这种方法可以检测过滤白蛋白整个鲍曼的空间（初始尿过滤室）的滤过屏障进入，也允许由近端肾小管重吸收白蛋白定量和随后的白蛋白transcytosis的6可视化。荧光白蛋白的情况下购买沿管状段途中膀胱突出检索通路在较早的近端小管段的效率。此外，当这种技术用于测定渗透率葡聚糖具有类似大小白蛋白几乎相同的渗透率值报告2。这些意见直接支持需要扩大许多蛋白尿的肾脏疾病的重点包括改变近端小管细胞填海的。

图3显示拍摄的图像，例如，从一个表面肾小球慕尼黑WistarFrömter的大鼠和所采取的步骤，以确定荧光蛋白的渗透性。 GSC的白蛋白的值0.0111来自这个人肾小球下降的慕尼黑Wistar大鼠的供给条件3时，该菌株在看到的范围内。在这些图像中看到的稳定性是由于仔细地规划和在图1和图2中所示的指令的执行。如前所述，切口的位置外肾是最关​​键的一步，在生产稳定?...

Watch this Scientific Journal Video about Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats at JoVE.com

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

利用高分辨率intavital 2 - 光子显微镜的技术直接可视化和量化表面肾小球过滤gloemrular。此方法允许在正常和疾病状态的大分子渗透特性的直接测定。

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Research

JoVE Journal

Medicine

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

科研

医学

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

一种使用加速度计量化上肢表现在日常生活的方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in MeniÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¨re's Disease Ears and Normal Hearing Ears

体内Menière 病耳和正常听觉的磁共振成像对人颅神经的形态学分析

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

小鼠肾小球滤过率的透皮测定

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

高灵敏度测量与氟素等西洋盐-聚糖小鼠的球状渗透性 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

小鼠窦房和房室结的全卡口免疫荧光染色、共聚焦成像和 3D 重建

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

小鼠窦房和房室结常驻心脏巨噬细胞的分离和培养

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

分析长期心电图记录以检测小鼠心律失常

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...