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

हम हमारे सभी साथियों को, जो इस परियोजना के विभिन्न चरणों में मदद, सलाह और महत्वपूर्ण इनपुट प्रदान शुक्रिया अदा करना चाहता हूँ. इस वीडियो लेख उत्पादन (IMIDIA से धन के साथ समर्थित किया गया <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ), जर्मन मधुमेह अनुसंधान के लिए केंद्र (DZD, शिक्षा और अनुसंधान (BMBF) के लिए जर्मन मंत्रालय <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd ev.de</a> ) और विश्वविद्यालय अस्पताल प्रौद्योगिकी ड्रेसडेन विश्वविद्यालय में कार्ल गुस्ताव सन्यास. इस प्रकाशन के लिए अग्रणी काम अनुदान समझौते के n ° 155,005 (IMIDIA), जो वित्तीय योगदान के यूरोपीय संघ के सातवें फ्रेमवर्क कार्यक्रम (FP7/2007-2013) और EFPIA से बना रहे हैं संसाधनों के तहत अभिनव दवाओं पहल संयुक्त उपक्रम से समर्थन प्राप्त हुआ है कंपनियों के तरह के योगदान में.

1. मानव अग्नाशय के ऊतकों की ठंड <ol><li> वसा ऊतकों, रक्त वाहिकाओं, नसों और गैर parenchymal एक स्केलपेल और चिमटी के साथ ऊतक निकालें, और टुकड़े (~ 0.5-1 सेमी cubes) में अग्नाशय के ऊतकों में कटौती. </li><li> एक Cryomold के केंद्र में एक अग्ना?...

<ol>
	<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>

हम मानव islets के लेजर (LMD) शल्य अग्नाशय नमूना से microdissection के लिए एक विश्वसनीय दृष्टिकोण का वर्णन. बशर्ते कि एक LMD माइक्रोस्कोप उपलब्ध है, इस प्रक्रिया में किसी भी शोध संस्थान में आंशिक pancreatectomies प्रदर्शन लागू किया ?...

<table border="1"><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां </td></tr><tr><td> 2-Methylbutane (isopentane) </td><td> ROTH </td><td> 3927.1 </td><td></td></tr><tr><td> AdhesiveCap (अपारदर्शी, 500 μl) </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td> Cellstar ट्यूबों (50 मिलीलीटर) </td><td> Greiner जैव - एक </td><td> २१० २६१ </td><td> समर्थन स्कर्ट के साथ </td></tr><tr><td> Cellstar ट्यूबों (50 मिलीलीटर) </td><td> Greiner जैव - एक </td><td> 227.261 </td><td></td></tr><tr><td> Diethyl pyrocarbonate (DEPC) </td><td> सिग्मा </td><td> 5758 डी </td><td></td></tr><tr><td> सूखी बर्फ </td><td></td><td></td><td></td></tr><tr><td> निरपेक्ष इथेनॉल </td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td> तरल नाइट्रोजन </td><td></td><td></td><td></td></tr><tr><td> ब्रश पेंट </td><td></td><td></td><td></td></tr><tr><td> पाम MicroBeam </td><td> ZEISS </td><td></td><td></td></tr><tr><td> पील एक मार्ग embedding (छोटा कर दिया) molds, 12 x 12 मिमी </td><td> ProSciTech </td><td> RR12 </td><td> शीर्ष आंतरिक 22x22 मिमी, 21 मिमी गहराई </td></tr><tr><td> Arcturus PicoPure जमे हुए शाही सेना अलगाव किट </td><td> एप्लाइड Biosystems </td><td> 0204 किट </td><td></td></tr><tr><td> प्लास्टिक क्लैंप </td><td></td><td></td><td></td></tr><tr><td> उस्तरा </td><td></td><td></td><td></td></tr><tr><td> RNase मुक्त DNase सेट </td><td> क्विएज़न </td><td> 79254 </td><td></td></tr><tr><td> RNaseZAP </td><td> सिग्मा </td><td> आर 2020 </td><td></td></tr><tr><td> / स्केल्पलसर्जिकल ब्लेड </td><td> टेक्नो कट </td><td> 2800111 </td><td></td></tr><tr><td> SuperFrost प्लस आसंजन खुर्दबीन स्लाइड </td><td> थर्मो वैज्ञानिक </td><td> J1800AMNZ </td><td> 25x75x1.0 मिमी </td></tr><tr><td> ऊतक टेक अक्टूबर यौगिक </td><td> Sakura </td><td> 4583 या 0807000022 </td><td></td></tr><tr><td> चिमटी </td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td> Xylene </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.

जैसा कि चित्र 1 में दिखाया गया है, dehydrating संशोधित प्रोटोकॉल पिछले प्रकाशित 6 प्रोटोकॉल की तुलना में बीटा कोशिकाओं autofluorescence के एक सुधार के लिए नेतृत्व किया. वर्णित प्रोटोकॉल लागू, 39 शल्य अग्नाशय नमू...

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

लेजर microdissection एक तकनीक है कि पैरेन्काइमा की मात्रा मिनट से चयनित कक्षों की वसूली की अनुमति देता है. यहाँ हम शल्य transcriptomic अध्ययन के लिए इस्तेमाल किया जा नमूनों से मानव अग्नाशय islets प्राप्त करने के लिए एक प्रोटोकॉल का वर्णन. हमारा प्रोटोकॉल मानव बीटा कोशिकाओं की आंतरिक autofluorescence, इस प्रकार उनके संग्रह की सुविधा में सुधार.

मानव अग्नाशय islets के सर्जिकल नमूनों से लेजर Microdissection में सुधार के लिए प्रोटोकॉल

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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