Name: isolation of glomeruli and in vivo labeling of glomerular cell surface proteins
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提交人感谢 blanka duvnjak 的出色技术援助。这项工作得到了德国妇女组织 (www.dfg.de) w1811\ 2-1 至 m. w. 和 qu2803-1 至智商的支持。资助者在研究设计、数据收集、数据分析、出版决定和手稿准备方面没有任何作用。

老鼠是从当地动物护理设施或法国 janvier 实验室获得的, 作为一种内部品种。调查是根据《实验动物护理和使用指南》 (美国国立卫生研究院85-23 出版物, 1996年修订) 中概述的准则进行的。所有动物实验都是按照相关机构批准进行的 (州政府 lanuv 参考编号 az:84-02.0 04)。2016. a435)。
1. 仪器、解决方案和设备的准备
<ol><li>制备1升磷酸盐缓冲盐水, 辅以 1 mm 氯化镁 (mgcl 2)和 0...

<ol>
	<li>Jefferson, J. A., Alpers, C. E., Shankland, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podocyte+biology+for+the+bedside.">Podocyte biology for the bedside.</a> American Journal of Kidney Disease. 58 (5), 835-845 (2011).</li><li>Konigshausen, E., 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=Angiotensin+II+increases+glomerular+permeability+by+beta-arrestin+mediated+nephrin+endocytosis.">Angiotensin II increases glomerular permeability by beta-arrestin mediated nephrin endocytosis.</a> Scientific Reports. 6, 39513 (2016).</li><li>Quack, I., 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=beta-Arrestin2+mediates+nephrin+endocytosis+and+impairs+slit+diaphragm+integrity.">beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity.</a> Proceedings of the National Academy of Science of the United States of America. 103 (38), 14110-14115 (2006).</li><li>Quack, I., 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=PKC+alpha+mediates+beta-arrestin2-dependent+nephrin+endocytosis+in+hyperglycemia.">PKC alpha mediates beta-arrestin2-dependent nephrin endocytosis in hyperglycemia.</a> Journal of Biological Chemitry. 286 (15), 12959-12970 (2011).</li><li>Swiatecka-Urban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+Trafficking+at+the+Mature+Podocyte+Slit+Diaphragm.">Endocytic Trafficking at the Mature Podocyte Slit Diaphragm.</a> Frontiers in Pediatrics. 5, 32 (2017).</li><li>Swiatecka-Urban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+trafficking+in+podocyte+health+and+disease.">Membrane trafficking in podocyte health and disease.</a> Pediatric Nephrology. 28 (9), 1723-1737 (2013).</li><li>Soda, K., 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=Role+of+dynamin,+synaptojanin,+and+endophilin+in+podocyte+foot+processes.">Role of dynamin, synaptojanin, and endophilin in podocyte foot processes.</a> The Journal of Clinical Investigation. 122 (12), 4401-4411 (2012).</li><li>Kestila, 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=Positionally+cloned+gene+for+a+novel+glomerular+protein--nephrin--is+mutated+in+congenital+nephrotic+syndrome.">Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome.</a> Molecular Cell. 1 (4), 575-582 (1998).</li><li>Martin, C. E., Jones, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrin+Signaling+in+the+Podocyte:+An+Updated+View+of+Signal+Regulation+at+the+Slit+Diaphragm+and+Beyond.">Nephrin Signaling in the Podocyte: An Updated View of Signal Regulation at the Slit Diaphragm and Beyond.</a> Frontiers in Endocrinology (Lausanne). 9, 302 (2018).</li><li>Nielsen, J. S., McNagny, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+podocalyxin+in+health+and+disease.">The role of podocalyxin in health and disease.</a> Journal of the American Society of Nephrology. 20 (8), 1669-1676 (2009).</li><li>Yasuda, T., Saegusa, C., Kamakura, S., Sumimoto, H., Fukuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab27+effector+Slp2-a+transports+the+apical+signaling+molecule+podocalyxin+to+the+apical+surface+of+MDCK+II+cells+and+regulates+claudin-2+expression.">Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression.</a> Molecular Biology of the Cell. 23 (16), 3229-3239 (2012).</li><li>Tossidou, I., 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=Podocytic+PKC-alpha+is+regulated+in+murine+and+human+diabetes+and+mediates+nephrin+endocytosis.">Podocytic PKC-alpha is regulated in murine and human diabetes and mediates nephrin endocytosis.</a> Public Library of Science One. 5 (4), 10185 (2010).</li><li>Qin, X. S., 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=Phosphorylation+of+nephrin+triggers+its+internalization+by+raft-mediated+endocytosis.">Phosphorylation of nephrin triggers its internalization by raft-mediated endocytosis.</a> Journal of the American Society of Nephrology. 20 (12), 2534-2545 (2009).</li><li>Waters, A. 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=Notch+promotes+dynamin-dependent+endocytosis+of+nephrin.">Notch promotes dynamin-dependent endocytosis of nephrin.</a> Journal of the American Society of Nephrology. 23 (1), 27-35 (2012).</li><li>Zhang, X., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+kinases+endocytosis+in+endothelium:+biology+and+signaling.">Receptor tyrosine kinases endocytosis in endothelium: biology and signaling.</a> Arteriosclerosis Thrombosis and Vascular Biology. 34 (9), 1831-1837 (2014).</li><li>Maes, H., Olmeda, D., Soengas, M. S., Agostinis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+trafficking+mechanisms+in+endothelial+cells+as+modulators+of+the+tumor+vasculature+and+targets+of+antiangiogenic+therapies.">Vesicular trafficking mechanisms in endothelial cells as modulators of the tumor vasculature and targets of antiangiogenic therapies.</a> Federation of European Biochemical Societies Journal. 283 (1), 25-38 (2016).</li><li>Satchell, S. C., 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=Conditionally+immortalized+human+glomerular+endothelial+cells+expressing+fenestrations+in+response+to+VEGF.">Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.</a> Kidney International. 69 (9), 1633-1640 (2006).</li><li>Takemoto, 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=A+new+method+for+large+scale+isolation+of+kidney+glomeruli+from+mice.">A new method for large scale isolation of kidney glomeruli from mice.</a> American Journal of Pathology. 161 (3), 799-805 (2002).</li><li>Liu, X., 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=Isolating+glomeruli+from+mice:+A+practical+approach+for+beginners.">Isolating glomeruli from mice: A practical approach for beginners.</a> Experimental and Therapeutic Medicine. 5 (5), 1322-1326 (2013).</li><li>Haase, R., 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=A+novel+in+vivo+method+to+quantify+slit+diaphragm+protein+abundance+in+murine+proteinuric+kidney+disease.">A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease.</a> Public Library of Science One. 12 (6), 0179217 (2017).</li><li>Satoh, 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=aPKClambda+maintains+the+integrity+of+the+glomerular+slit+diaphragm+through+trafficking+of+nephrin+to+the+cell+surface.">aPKClambda maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface.</a> Journal of Biochemistry. 156 (2), 115-128 (2014).</li><li>Tomas, N. 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=Thrombospondin+type-1+domain-containing+7A+in+idiopathic+membranous+nephropathy.">Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy.</a> New England Journal of Medicine. 371 (24), 2277-2287 (2014).</li><li>Daniels, G. M., Amara, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+neurotransmitter+transporters+at+the+cell+surface.">Selective labeling of neurotransmitter transporters at the cell surface.</a> Methods in Enzymology. 296, 307-318 (1998).</li><li>Ougaard, M. K. E., 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=Murine+Nephrotoxic+Nephritis+as+a+Model+of+Chronic+Kidney+Disease.">Murine Nephrotoxic Nephritis as a Model of Chronic Kidney Disease.</a> International Journal of Nephrology. 2018, 8424502 (2018).</li><li>Salant, D. J., Darby, C., Couser, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+membranous+glomerulonephritis+in+rats.+Quantitative+studies+of+glomerular+immune+deposit+formation+in+isolated+glomeruli+and+whole+animals.">Experimental membranous glomerulonephritis in rats. Quantitative studies of glomerular immune deposit formation in isolated glomeruli and whole animals.</a> Journal of Clinical Investigation. 66 (1), 71-81 (1980).</li></ol>

该方法可以成功地分离肾小球, 以研究肾小球 rna 或蛋白质。此外, 原代肾小球细胞培养可以从分离的肾小球进行。如果生物素是在肾小球分离之前应用的, 则可以对肾小球细胞表面蛋白进行标记。利用该方法可以研究体内肾小球细胞表面蛋白的贩运, 并可对蛋白质丰度进行半定量。成功检测肾小球细胞表面蛋白丰度的最关键步骤是: 1) 发展小鼠手术方面的手工专业知识, 特别是主动脉插管, 2) ...

蛋白尿是肾小球损伤的标志, 通常伴随肾小球滤池 1的破坏。肾小球滤池由被过滤的内皮细胞、肾小球基底膜和足细胞组成。肾小球滤池的微妙分子结构是高度动态的, 在健康和患病的肾脏2,3,4, 5, 6 都容易发生细胞表面蛋白贩运.细胞表面蛋白的内分泌已被证明是至关重要的生存的足细胞7。肾素和足苷酸是在足细胞上表达的跨膜蛋白。肾素是肾小球裂隙膜片的主干, 而 podocalyxin 是一种四聚糖蛋白, 包覆于足部 8、9、10 的次生足部过程。此前, 已经显示肾素和 podocalyxin3、11、12、13、14的内分泌贩运。
据我们所知, 细胞表面蛋白的内吞尚未在肾小球内皮细胞中被描述在文献中。然而, 内皮细胞一般表达所有必要的蛋白质为不同类型的内吞 (即, clathrin 依赖, 流依赖性内吞)15,16。因此, 可以用这种方法研究内皮细胞表面的移植, 例如, 使用血管内皮 (ve)-钙粘蛋白和细胞内粘附分子 (icam-2) 作为肾小球内皮细胞17的细胞表面标记蛋白.
遗憾的是, 对于可以研究细胞表面蛋白质贩运的精细三层肾小球滤池, 目前还没有准确的体外模型。因此, 这种方法的目的是研究体内肾小球蛋白贩运。此外, 该协议还包含有关如何分离肾小球的信息, 从而能够进一步分析肾小球 rna、蛋白质或细胞。不同的组 18,19描述了类似的肾小球分离技术。
此前, 我们和其他人使用了生物素化2、3、4、20、21对肾小球细胞表面蛋白进行体外标记。然而, 在这种体外方法中, 孤立的肾小球暴露在机械应力下, 这可能会影响细胞内贩运。另外, 肾小球细胞表面蛋白的免疫荧光标记已被广泛应用于文献2、20、22.然而, 使用这种方法, 只有少量的蛋白质可以在一张幻灯片内进行分析, 并且免疫荧光图像的定量往往是困难的。
这种新的体内方法为研究健康和患病肾脏中肾小球细胞表面蛋白质丰度和贩运提供了可靠的工具, 可作为免疫荧光检测的补充。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Motic SMZ168 BL</td>
			<td style="width:77px;">Motic</td>
			<td style="width:101px;">SMZ168BL</td>
			<td style="width:235px;">microscope for mouse surgery</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:257px;">KL1500LCD</td>
			<td style="width:77px;">Pulch and Lorenz microscopy</td>
			<td style="width:101px;">150500</td>
			<td>light for mouse surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td>PZN:01320422</td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Microfederschere</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">FD100R</td>
			<td>fine scissors, for cut into the aorta</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Durotip Feine Scheren</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BC210R</td>
			<td>for abdominal cut</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Anatomische Pinzette</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BD215R</td>
			<td>for surgery until the abdomen is opened</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Pr&#228;parierklemme</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">BJ008R</td>
			<td>for surgery&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Seraflex</td>
			<td style="width:77px;">Serag Wiessner</td>
			<td style="width:101px;">IC108000</td>
			<td>silk thread</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Ketamine 10%</td>
			<td style="width:77px;">Medistar</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/100</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/200</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Harvard apparatus 11 Plus</td>
			<td style="width:77px;">Harvard Apparatus</td>
			<td style="width:101px;">70-2209</td>
			<td>syringe pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">EZ-link Sulfo-NHC-LC-Biotin</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">21335</td>
			<td>biotin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Dynabeads Untouched Mouse T-cells</td>
			<td style="width:77px;">Invitrogen</td>
			<td style="width:101px;">11413D</td>
			<td>to embolize glomeruli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Collagenase A</td>
			<td style="width:77px;">Roche</td>
			<td style="width:101px;">10103578001</td>
			<td>to digest kidney tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DynaMag-2</td>
			<td style="width:77px;">Invitrogen</td>
			<td style="width:101px;">123.21D</td>
			<td>Magnet catcher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">100&#181;m cell stainer</td>
			<td style="width:77px;">Greiner-bio</td>
			<td style="width:101px;">542000</td>
			<td>for glomerular isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Axiovert 40 CFL</td>
			<td style="width:77px;">Zeiss</td>
			<td style="width:101px;">non available</td>
			<td>to confirm glomerular purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">TissueRuptor</td>
			<td style="width:77px;">Quiagen</td>
			<td style="width:101px;">9002755</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">CHAPS</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">C3023</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Tris-HCL</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">T5941</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">NaCl</td>
			<td style="width:77px;">VWR chemicals</td>
			<td style="width:101px;">27810295</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">NaF</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">201154</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">EDTA</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">E5134</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">ATP</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">34369-07-8</td>
			<td>for lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">23225</td>
			<td>Follow the manufacturer&#39;s instructions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">nephrin antibody</td>
			<td style="width:77px;">Progen</td>
			<td style="width:101px;">GP-N2</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Polyclonal goat anti-podocalyxin antibody</td>
			<td style="width:77px;">R&#38;D Systems</td>
			<td style="width:101px;">AF15556-SP</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Streptavidin Agarose Resin</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">20347</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Protein A sepharose CL-4B</td>
			<td style="width:77px;">GE Healthcare</td>
			<td style="width:101px;">17096303</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">polyclonal rabbit anti-p57 antibody</td>
			<td style="width:77px;">SCBT</td>
			<td style="width:101px;">sc-8298</td>
			<td>for Immunohistochemistry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">mouse monoclonal anti-beta actin antibody, clone AC-74</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2228</td>
			<td>Western blot loading control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">rabbit anti-p44/42</td>
			<td style="width:77px;">cell signalling</td>
			<td style="width:101px;">4695</td>
			<td>for westernblot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Pierce High sensitivity streptavidin-HRP</td>
			<td style="width:77px;">Thermo Scientific</td>
			<td style="width:101px;">21130</td>
			<td>for westernblot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyclonal mouse ICAM-2 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF774</td>
			<td>for westernblot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyclonal mouse anti-VE-cadherin</td>
			<td>R&#38;D Systems</td>
			<td>AF1002</td>
			<td>for westernblot</td>
		</tr>
	</tbody>
</table>

isolation of glomeruli and in vivo labeling of glomerular cell surface proteins

蛋白尿是由被过滤的内皮细胞、肾小球基底膜和带缝隙膜片组成的肾小球滤池的破坏引起的。肾小球滤池的微妙结构, 特别是裂隙膜片, 依赖于不同细胞表面蛋白质的相互作用。到目前为止, 对这些细胞表面蛋白的研究仅限于体外研究或组织学分析。在这里, 我们提出了一个小鼠体内生物素化标记方法, 使我们能够研究肾小球细胞表面蛋白在生理和病理生理条件下。该协议包含有关如何灌注小鼠肾脏, 分离肾小球, 并执行内源性免疫沉淀的感兴趣的蛋白质的信息。这种新方法可以对肾小球细胞表面丰度进行半定量, 并可研究生物素灌注和免疫沉淀所能获得的所有蛋白质。此外, 通过在生物素化或不生物素化的情况下分离肾小球, 可以进一步分析肾小球 rna 和蛋白质以及原代肾小球细胞培养 (即原代肾小球细胞培养)。

为了准确地分离肾小球, 必须先用 pbscm 灌注小鼠肾脏。pbscm 灌注使肾脏变白 (图 1a)。用磁珠栓塞肾小球将可见于肾脏表面的棕色圆点 (图 1b)。用捕磁器分离肾小球可能显示肾小管污染 (图 1c)。在进一步分析肾小球之前, 需要通过更彻底地清洗肾小球来达到 > 95% 的肾小?...

Watch this Scientific Journal Video about Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins at JoVE.com

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

在这里, 我们提出了一个协议的小鼠体内标记肾小球细胞表面蛋白与生物素.该协议包含有关如何灌注小鼠肾脏, 分离肾小球, 并执行内源性免疫沉淀的感兴趣的蛋白质的信息。

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

该方法有助于回答肾病学领域有关细胞表面蛋白贩运在蛋白尿中作用的关键问题，而不是蛋白尿肾病。该技术的主要优点是，它有利于细胞表面蛋白质在体内的贩运研究，并且每个实验可以分析一种以上蛋白质。虽然这种方法可以提供对球状细胞表面贩运的见解，也可以应用于其他器官系统，这些器官系统被移植，并且可以通过隔离技术获得。首先确认对头趾捏没有反应，用70%异丙基对麻醉小鼠的腹侧进行消毒。使中位切入皮肤从骨盆到胸骨，并使用钳子去除腹部筋膜的皮肤。切开腹部中线两侧的皮肤，并通过腹部肌肉层从膀胱到西腓的介质。使用剪刀和钳子将肌肉分成四个象限，然后使用两个手术钳将上两个象限固定到动物的颈部。将动物放在解剖显微镜下，使用无菌交换将内脏器官移出。用细剪刀，切下肝性韧带。使用精细的手术钳子，在肾动脉的肝肠动脉周围放置一个缝合线。在主动脉周围，与肾动脉近在近，肾上腺。将远大肌从筋膜、脂肪和其他组织中释放。然后，在分叉的高度夹紧静脉和主动脉，并在肾动脉主动脉周围放置另一个缝合线。使用钳子拧紧近角主动脉缝合，衰减血流，将主动脉直径的一半的小孔切入主动脉，向肾动脉开去。将一个10厘米的导管插入附在10毫升注射器上，注射器中含有5毫升的冰冷PBS加上钙和镁。用准备好的连字固定导管。然后开始以每分钟两毫升的流速对肾脏进行足衰竭。使用细剪刀在肾动脉的肾静脉中切一个洞，并收紧肝动脉和肠动脉周围的缝合线。交付整个 PBS 体积后，用五毫升冰冷 PBS 加上钙和镁代替香水，并辅以每毫升 0.5 毫克的生物素进行表面标记。继续以每分钟两毫升的流速使肾脏保持领先。当整个体积已交付时，将含有五毫升冰冷 PBS 的注射器以及钙和镁，并辅以 100 毫升甘氨酸到导管上。以每分钟两毫升的流速，用甘氨酸溶液的所有五毫升来淬火。然后用五毫升的冰冷PBS加上钙和镁补充肾脏，每毫升200微升磁珠，每分钟流速为2毫升。用棕色磁珠的球状栓塞将在肾脏表面可见。当所有的珠子已经交付删除肾脏的胶囊和收获肾脏在欣隆。将肾脏放在含有 15 毫升 PBS 加钙和镁的 10 厘米细胞培养盘中。使用新的双刃刀片将肾脏切成尽可能小的碎片。将组织转移到含有一毫升胶原酶 A 的两毫升管中，在 37 摄氏度下进行 30 分钟，在消化前和消化后用经过修改的 1000 微升移液器尖端轻轻混合组织溶液。在孵育结束时，通过100微米细胞过滤器过滤消化的组织。使用细胞刮刀和冰冷PBS加上钙和镁，通过细胞过滤器捣碎剩余的组织。将肾单细胞悬浮液的最终体积与新鲜冰冷PBS加钙和镁一起带到50毫升，通过离心收集肾细胞。取出上清液，然后用略低于1.5毫升的冰冷PBS加钙和镁重新悬浮颗粒，同时涡旋并转移球状悬浮液到新的两毫升管中。要清洗球状，请将管子放入磁铁中，以聚合球状体。一分钟后，使用 1000 微升移液器取出上流液。从磁铁上取下管子。在组织中加入一毫升的 PBS 以及钙和镁。移液球状上下一次，并漩涡细胞。将管子返回磁铁，然后再次清洗球状，正如刚刚演示的。直到样品纯度达到至少 90%的光显微镜分析在 40 至 100 X 放大倍率。然后通过离心收集磁珠纯化的光泽。为了达到超过 95% 的纯度，需要彻底清洗，如证明。生物素灌注有助于在生物素中标记毛细管环，但不受控制的灌注小鼠肾脏。球状提取物生物素部分的免疫沉淀表明，肾上腺素和足腺素是免疫沉淀的生物素，但不是与控制分数。免疫沉淀分数与斯特雷普他丁的染色进一步证实了生物素在生物素中所检发的内腓林的存在，但无法控制被注入的动物。内皮蛋白血管内皮球蛋白和细胞内粘附分子二也是免疫沉淀的生物素分数。在肾毒性肾炎的早期，与对症相比，肾毒性肾炎动物观察到细胞表面肾素的表达减少。事实上，密度分析显示，与控制者相比，肾毒性性肾炎动物的生物素甲酸肾上腺素显著减少。对肾素与作用素总比例的定量分析表明，对照组与肾毒性肾炎小鼠之间没有显著差异。此外，在肾毒性肾炎和控制动物中观察到同样数量的豆瓣细胞。在晚期肾毒性肾炎中，肾毒性性肾毒动物表现出肾素表达的恢复，对照组和肾毒性性肾毒小鼠之间测量的细胞表面肾素无显著差异，肾毒性肾毒动物的整体肾素减少。此外，与控制动物相比，豆瓣细胞数量减少。在尝试此程序时，重要的是要记住，保持注射器在连接过程中无气泡，以避免球状体的空气栓塞，并在肾脏灌注开始后在冰冷的条件下工作。按照这个程序，其他方法，如分离球状RNA和蛋白质可以回答有关蛋白质表达或相互作用在健康和患病肾脏的其他问题。

Research

JoVE Journal

Biochemistry

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

科研

生物化学

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

极快速和特殊的代谢标签的RNA在Vivo与4-Thiouracil （Ers4tU）

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...