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>
2. Preparation of Frozen Sections
<ol>
 <li>Wear gloves throughout all steps to avoid denaturation of RNA. </li>
 <li>Clean the cryostat knife, specimen holder and paintbrush with cold 100% ethanol and RNase Away. Place the frozen tissue inside the cryostat. </li>
 <li>Wait until the tissue, knife and brush reach -20 &deg;C. Label the SuperFrost Plus slides and pre-cool them inside the cryostat chamber while waiting for the tissue to reach -20 &deg;C. </li>
 <li>Apply the Tissue-Tek O.C.T. Compound on the specimen holder and place the frozen tissue on top. </li>
 <li>Once the Tissue-Tek O.C.T. Compound is frozen trim the cryoblock and remove any excess of Tissue-Tek O.C.T. Compound with a razor blade. </li>
 <li>Cut a total of 40 consecutive 10 &mu;m slices from the pancreatic tissue block. Additionally we recommend cutting a first and middle slice to be saved for overview drawings of the pancreatic section that can facilitate the identification of islets within the slices during the microdissection process. </li>
 <li>Transfer the sections from the knife blade to the center of a SuperFrost Plus slide by touching the backside of the slide with your finger (covered with a glove) to warm up only the region to which the section shall adhere. </li>
 <li>Dry the sections 1-2 min inside the cryostat at -20 &deg;C, after which you put them into a slide box placed on dry ice. Store the sections at -80 &deg;C. </li>
 <li>If necessary, clean the knife blade with paper towels and cold 100% ethanol. </li>
</ol>
3. Dehydration of Frozen Sections 
<ol>
 <li>Prepare the Reagents: pour 30 ml freshly prepared 70% ethanol, 30 ml DEPC-treated water, 2x30 ml 100% ethanol and 30 ml xylene into 50 ml Cellstar tubes (Falcons); chill and keep all reagents (except xylene) on ice. </li>
 <li>Clean the working space under the hood with 100% ethanol and RNaseZAP. </li>
 <li>Process two cryosections as follows: fix in ice cold 70% ethanol for 30 sec, wash with 5-6 quick dips in ice cold DEPC-treated water, dehydrate twice for 1 min in ice cold 100% ethanol, incubate for 4 min in xylene at room temperature (25 &deg;C), and air dry for 3-5 min. Repeat this step with another pair of cryosections. </li>
 <li>Insert two dried cryosections back to back into a 50 ml Cellstar tube wrapped with aluminium foil to protect them from light and moisture. </li>
</ol>
4. Laser Microdissection of Beta-cells with PALM MicroBeam, Zeiss
<ol>
 <li>Clean the microscope with RNaseZAP and mount the Adhesive Cap in the RoboMover.</li>
 <li>Set the following settings: Filter set 09 (excitation: 450-490 nm, emission &gt; 515 nm), AutoLPC mode, 50% laser energy, 65% laser focus, 100% laser speed, 5 &mu;m distance between AutoLPC shots, 6 &mu;m distance to line, working height: -10,100.</li>
 <li>Insert one slide into the stage. </li>
 <li>Locate the autofluorescent beta cells under microscopic visualisation (5x or 10x lens).</li>
 <li>Switch to the 40x lens, select the beta cells with the "Freehand" selection tool and microdissect them using the laser. </li>
 <li>Capture the tissue of four dehydrated cryosections into one AdhesiveCap. 
 
 Comment 1: the time to dissect the beta cells of one dehydrated cryosection should not be longer than 10-20 min to avoid rehydration of the tissue sections which would result in RNA degradation. 
 Comment 2: verify successful microdissection process by visual inspection after moving the stage to the checkpoint. </li>
</ol>
5. Lysis of Microdissected Beta Cell Enriched Tissue 
<ol>
 <li>Remove the AdhesiveCap from the RoboMover. </li>
 <li>Pipet 10 &mu;l extraction buffer (XB, PicoPure RNA Isolation Kit, Arcturus) into the lid of the AdhesiveCap and incubate it upside down at 42 &deg;C for 30 min. </li>
 <li>Spin down at 10,000 x g and put the lysate on dry ice. Store it at -80 &deg;C until the RNA extraction is performed. </li>
 <li>Repeat steps 3.) to 5.) with all other sections. </li>
</ol>
6. RNA Extraction 
<ol>
 <li>Combine all ten 10 &mu;l lysates </li>
 <li>Proceed with the RNA isolation by working at room temperature according to the protocol of the PicoPure RNA Isolation Kit, Arcturus, using a 1:1 ratio Extraction Buffer (XB) to 70% Ethanol (including DNase Treatment). </li>
</ol>
7. RNA Analysis 
<ol>
 <li>Use 1 &mu;l RNA for analysis of quality and quantity using an Agilent 2100 Bioanalyser (PicoChip). Quantitative evaluation is done in reference to a standard RNA loaded in parallel on the chip. </li>
</ol>

<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>

No conflicts of interest declared.

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...

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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 ...

improved-protocol-for-laser-microdissection-human-pancreatic-islets

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14.3K Views.  Paul Langerhans Institute Dresden. 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.