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

We want to thank all our colleagues who provided help, advice and critical input at various steps of this project. Production of this video article was supported with funds from IMIDIA (<a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a>), the German Ministry for Education and Research (BMBF) to the German Centre for Diabetes Research (DZD, <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd-ev.de</a>) and the University Hospital Carl Gustav Carus at the University of Technology Dresden. The work leading to this publication has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n &deg; 155005 (IMIDIA), resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution.

1. Freezing of Human Pancreatic Tissue
<ol>
 <li>Remove adipose tissue, blood vessels, nerves and non-parenchymal tissue with a scalpel and tweezers, and cut the pancreatic tissue into pieces (~0.5-1 cm cubes). </li>
 <li>Place one pancreatic tissue piece in the center of a Cryomold, and cover it completely with cold Tissue-Tek O.C.T. Compound. Put the Cryomold into a jar whose bottom is covered with pre-cooled 2-Methylbutane and snap-freeze in liquid nitrogen. Store the frozen sample at -80 &deg;C. </li>
</ol>
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<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>

We describe a reliable approach for the laser microdissection (LMD) of human islets from surgical pancreatic specimen. Provided that a LMD microscope is available, this procedure could be implemented at any research institution performing partial pancreatectomies, thereby increasing access to human islet material from both non-diabetic and type 2 diabetic subjects. This is especially relevant given the paucity of pancreata offered for islet isolation. Favourable aspects of LMD compared to islet isolation by collagenase d...

<table border="1">
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>2-Methylbutane 			(isopentane)</td>
 <td>ROTH</td>
 <td>3927.1</td>
 <td></td>
 </tr>
 <tr>
 <td>AdhesiveCap (opaque, 			500 &mu;l)</td>
 <td>ZEISS</td>
 <td>415190-9201-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Cellstar 			Tubes (50 ml)</td>
 <td>greiner 			bio-one</td>
 <td>210 			261</td>
 <td>with 			support skirt</td>
 </tr>
 <tr>
 <td>Cellstar 			Tubes (50 ml)</td>
 <td>greiner 			bio-one</td>
 <td>227.261</td>
 <td></td>
 </tr>
 <tr>
 <td>Diethyl 			pyrocarbonate (DEPC)</td>
 <td>SIGMA</td>
 <td>D 			5758</td>
 <td></td>
 </tr>
 <tr>
 <td>Dry 			ice</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol 			absolute</td>
 <td>VWR</td>
 <td>20821.310</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid 			nitrogen</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint 			brush</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 			embedding moulds (truncated), 12 x 12 mm</td>
 <td>ProSciTech</td>
 <td>RR12</td>
 <td>Top 			internal 22x22 mm, depth 21 mm</td>
 </tr>
 <tr>
 <td>Arcturus PicoPure 			Frozen RNA Isolation Kit</td>
 <td>Applied 			Biosystems</td>
 <td>KIT 			0204</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 			clamp</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Razor</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RNase-Free 			DNase 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>Scalpel 			/ surgical blade</td>
 <td>Techno 			Cut</td>
 <td>2800111</td>
 <td></td>
 </tr>
 <tr>
 <td>SuperFrost 			Plus adhesion microscope slides</td>
 <td>Thermo 			Scientific</td>
 <td>J1800AMNZ</td>
 <td>25x75x1.0 mm</td>
 </tr>
 <tr>
 <td>Tissue-Tek 			O.C.T Compound</td>
 <td>Sakura</td>
 <td>4583 			or 0807000022</td>
 <td></td>
 </tr>
 <tr>
 <td>Tweezers</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.

As shown in Figure 1, the modified dehydrating protocol led to an improvement of beta cells autofluorescence compared to the previous published protocol 6. Applying the described protocol, each of 39 surgical pancreatic specimens was used to generate 40 serial cryosections for an average of 31'544'704 &mu;m3 tissue/ pancreatic specimen (range: 8'742'390 - 81'522'153 &mu;m3) as shown in Table 1. This represents the volume of about 18 pancreatic islets of...

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

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

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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