Name: improved protocol for laser microdissection of human pancreatic islets from surgical specimens
Uploaded: 2013-01-06T14:00:00
Description: Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

Мы хотим поблагодарить всех наших коллег, которые предоставили помощь, советы и критические входа на различных этапах этого проекта. Производство этого видео статьи была поддержана за счет средств IMIDIA ( <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ), немецкого министерства образования и научных исследований (BMBF) к немецким центром исследований диабета (DZD, <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd -ev.de</a> ) и Университетской больницы Карл Густав Карус в Технологическом университете Дрездена. Работы, ведущей к данной публикации получило поддержку со стороны Инновационные инициативы лекарственных средств совместного проекта по гранту соглашения N ° 155005 (IMIDIA), ресурсы которого состоят из финансового вклада Седьмой рамочной Европейского Союза, Программы (FP7/2007-2013) и EFPIA компаний в натуральном выражении.

1. Замораживание человеческих тканей поджелудочной железы <ol><li> Удалить жировой ткани, кровеносные сосуды, нервы и не паренхиматозной ткани с помощью скальпеля и пинцета, и сократить ткани поджелудочной железы на куски (~ 0.5-1 кубов см). </li><li> Поместите один кусок ткани поджелудочной жел?...

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	<li>Bonner, R. F., Emmert-Buck, 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Suarez-Quian, C. A., Goldstein, S. 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=Laser+capture+microdissection+of+single+cells+from+complex+tissues.">Laser capture microdissection of single cells from complex tissues.</a> Biotechniques. 26 (2), 328-3235 (1999).</li><li>Espina, V., Wulfkuhle, J. 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1, 586-603 (2006).</li><li>Mikulowska-Mennis, A., Taylor, T. B., 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=High-quality+RNA+from+cells+isolated+by+laser+capture+microdissection.">High-quality RNA from cells isolated by laser capture microdissection.</a> Biotechniques. 33 (1), 176-179 (2002).</li><li>B&#246;tticher, G., Sturm, D., Ehehalt, F., Knoch, K. P., Kersting, S., Gr&#252;tzmann, 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=Isolation+of+Human+Islets+from+Partially+Pancreatectomized+Patients.">Isolation of Human Islets from Partially Pancreatectomized Patients.</a> J. Vis. Exp. (53), e2962 (2011).</li><li>Marselli, L., Sgroi, D. 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=Laser+capture+microdissection+of+human+pancreatic+beta-cells+and+RNA+preparation+for+gene+expression+profiling.">Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.</a> Methods in Molecular Biology. 560, 87-98 (2009).</li><li>Marselli, L., Thorne, J., 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=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clinical Endocrinology &amp; Metabolism. 93, 1046-1053 (2008).</li><li>Bottino, R., Balamurugan, A. N., 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=Response+of+human+islets+to+isolation+stress+and+the+effect+of+antioxidant+treatment.">Response of human islets to isolation stress and the effect of antioxidant treatment.</a> Diabetes. 53 (10), 2559-2568 (2004).</li><li>Abdelli, S., Ansite, J., 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=Intracellular+stress+signaling+pathways+activated+during+human+islet+preparation+and+following+acute+cytokine+exposure.">Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.</a> Diabetes. 53 (11), 2815-2823 (2004).</li><li>Negi, S., Jetha, A., 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=Analysis+of+beta-cell+gene+expression+reveals+inflammatory+signaling+and+evidence+of+dedifferentiation+following+human+islet+isolation+and+culture.">Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.</a> PLoS One. 7 (1), e30415 (2012).</li></ol>

Мы описываем надежный подход для лазерных микродиссекции (LMD) человеческих островков от хирургического образца поджелудочной железы. При условии, что LMD микроскопа доступно, эта процедура может быть реализована в любой научно-исследовательского учреждения выполнения частичного pancreate...

<table border="1"><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии </td></tr><tr><td> 2-метилбутан (изопентан) </td><td> ROTH </td><td> 3927,1 </td><td></td></tr><tr><td> AdhesiveCap (непрозрачный, 500 мкл) </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td> Cellstar труб (50 мл) </td><td> Greiner Bio-One </td><td> 210 261 </td><td> с поддержкой юбки </td></tr><tr><td> Cellstar труб (50 мл) </td><td> Greiner Bio-One </td><td> 227,261 </td><td></td></tr><tr><td> Диэтиловый pyrocarbonate (DEPC) </td><td> SIGMA </td><td> D 5758 </td><td></td></tr><tr><td> Сухой лед </td><td></td><td></td><td></td></tr><tr><td> Этанол абсолютная </td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td> Жидкий азот </td><td></td><td></td><td></td></tr><tr><td> Кисть </td><td></td><td></td><td></td></tr><tr><td> PALM Microbeam </td><td> ZEISS </td><td></td><td></td></tr><tr><td> Peel-A-Way вложение форм (усеченный), 12 х 12 мм </td><td> ProSciTech </td><td> RR12 </td><td> Самые активные 22x22 мм, глубина 21 мм </td></tr><tr><td> Арктур ​​PicoPure Frozen выделения РНК Kit </td><td> Applied Biosystems </td><td> KIT 0204 </td><td></td></tr><tr><td> Зажим </td><td></td><td></td><td></td></tr><tr><td> Бритва </td><td></td><td></td><td></td></tr><tr><td> РНКазы ДНКазой Set </td><td> Qiagen </td><td> 79254 </td><td></td></tr><tr><td> RNaseZAP </td><td> SIGMA </td><td> R 2020 </td><td></td></tr><tr><td> Скальпель /хирургические лезвия </td><td> Techno Cut </td><td> 2800111 </td><td></td></tr><tr><td> SuperFrost Plus слайды адгезии микроскопом </td><td> Thermo Scientific </td><td> J1800AMNZ </td><td> 25x75x1.0 мм </td></tr><tr><td> Tissue-Tek октября соединение </td><td> Сакура </td><td> 4583 или 0807000022 </td><td></td></tr><tr><td> Пинцет </td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td> Ксилол </td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

improved protocol for laser microdissection of human pancreatic islets from surgical specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Как показано на рисунке 1, модифицированный протокол обезвоживания привели к улучшению бета автофлуоресценции клеток по сравнению с предыдущим опубликован протокол 6. Применение описанного протокола, каждый из 39 хирургических образцах поджелудочной железы был использ...

Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Лазерная микродиссекции является метод, который позволяет восстановление выделенных ячеек незначительное количество паренхимы. Здесь мы опишем протокол для приобретения человеческих панкреатических островков из операционного материала, который будет использоваться для транскриптомных исследований. Наш протокол улучшает внутреннее аутофлюоресценции человека бета-клетки, способствуя тем самым их коллекции.

Улучшенный протокол для лазерных микродиссекции человека панкреатических островков от хирургических образцах

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

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