Name: pancreatic islet embedding for paraffin sections
Uploaded: 2018-06-29T14:30:00
Description: Watch this Scientific Journal Video about Pancreatic Islet Embedding for Paraffin Sections at JoVE.com

We gratefully acknowledge the Beta Cell Biology Group at the UMass Diabetes Center of Excellence for helpful advice and discussions. Human pancreatic islets were provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope. This work was funded by NIH/NIDDK: R01-DK114686 (LCA), R01-DK105837 (CY), 2UC4DK098085 (IIDP) and by the American Diabetes Association grant #1-15-BS-003 (LCA) in collaboration with the Order of the Amaranth.

All procedures involving animals were approved by the UMass Medical School Institutional Animal Care and Use Committee. Human islet studies were determined by the UMass Institutional Review Board to not qualify for IRB review or exemption because they do not involve the use of human subjects.
1. Islet Isolation and Culture
<ol>
	<li>Isolate islets and separate from contaminating exocrine and ductal tissue using the method of your choice. 
	NOTE: This method was optimized using islets is...

<ol>
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L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocols+for+human+pancreatic+islets.">Staining protocols for human pancreatic islets.</a> Journal of Visualized Experiments. (63), (2012).</li><li>Da Silva Xavier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cells+of+the+Islets+of+Langerhans.">The Cells of the Islets of Langerhans.</a> Journal of Clinical Medicine. 7 (3), (2018).</li><li>Sharma, R. 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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=Novel+observations+from+next-generation+rna+sequencing+of+highly+purified+human+adult+and+fetal+islet+cell+subsets.">Novel observations from next-generation rna sequencing of highly purified human adult and fetal islet cell subsets.</a> Diabetes. 64 (9), 3172-3181 (2015).</li><li>Brissova, 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=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> Journal of Histochemistry &amp; Cytochemistry. 53 (9), 1087-1097 (2005).</li><li>Joiner, K. S., Spangler, E. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+beta-cell+proliferation+and+retinoblastoma+protein+phosphorylation+in+rat+and+human+islets+using+adenovirus-mediated+transfer+of+cyclin-dependent+kinase-4+and+cyclin+D1.">Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.</a> Diabetes. 53 (1), 149-159 (2004).</li><li>La Fortune, K. A., Randolph, M. L., Wu, H. H., Cramer, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+cell+block+processing:+The+Cell-Gel+method.">Improvements in cell block processing: The Cell-Gel method.</a> Cancer Cytopathology. 125 (4), 267-276 (2017).</li><li>Alonso, L. 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This modified gel-disc-based embedding method provides a simple, inexpensive, and efficient way to generate a high yield of islets per section. Constructing the gel disc on a flat glass surface facilitates spreading islets in an even distribution over a well-defined area. Spreading the islets in a flat disc offers the advantage of placing many islets in the plane of section, optimizing yield and allowing fewer islets to be used. The disc thickness can be adjusted to meet investigators&#39; needs. Since multiple sections ...

Pancreatic islets of Langerhans, the sole source of circulating insulin, are a critical tissue for investigators studying diabetes mellitus. From any given organism, islets have variable size, cell type frequency, and architecture1,2,3. The conventional strategy to study in vivo structure and endocrine cell composition of pancreatic islets is by sectioning pancreas tissue4,5. Since islets comprise only a small fraction of total pancreatic cellular content, molecular studies are performed on isolated islets. Ex vivo islet culture experiments testing response to nutrients, gene modulation (transfection, transduction), or experimental treatments provide important insight into mechanisms modulating endocrine cell survival, proliferation, and function5,6.
Ex vivo islet experiments often are analyzed using molecular studies of whole islets or histological or molecular studies of dispersed islet cells grown in monolayer5,6. Molecular analysis of whole islets introduces the serious caveat of intermixing cell types, which may produce false-negative or false-positive results when extrapolated to any individual cell type. Cell dispersion onto coverslips for post-culture microscopy allows cell-type-specific outcome measurement, but disrupts islet architecture, which may alter response to intervention and precludes identification of architecture-related outcomes. In addition, generally only a single outcome can be measured; for example, to measure beta cell proliferation and beta cell death under the same conditions, two separate experiments need to be performed. These approaches are also blind to inter-islet variability, an area of increasing interest in the field. Sorting islet cells by flow cytometry for cell-type-specific molecular studies, or single-cell RNA studies are elegant but expensive, time consuming, limited by tissue abundance, architecture-eradicating, and not well suited to routine cell culture analyses5,7. Confocal imaging of whole-mount immunostained islets provides high-quality intact-architecture data, but is labor intensive, and data obtained from each sample is limited to outcomes identifiable in a single immunostaining8.
The ability to generate high-quality paraffin sections of post-culture whole islets would address many of these concerns. High-cost, low-abundance islet tissue from unique genetic models or from human organ donors, or islets status post in vivo or in vitro experimental manipulation, are precious. Obtaining multiple paraffin sections from the same islets would allow multiple cell-type-specific, intact-architecture analyses from the same experiment.
Existing techniques to generate islet pellets for sectioning are imperfect. Histology-optimized agarose is an aqueous low melting point gel that is widely used in processing histological and cytological specimens including small or fragmented tissue samples that are difficult to process9. One islet embedding approach is to suspend the islets in agarose in a microcentrifuge tube, centrifuge to pellet the material, retrieve the agar plug, then process and embed for sectioning10,11. Extracting the solidified sample from the bottom of the tube is time consuming and difficult, leading to occasional fragmentation of the sample and risk of personal injury. The islets are concentrated in the tip of the plug, leading to inadequate islet distribution in sections obtained from this method. The round bottom of the plug complicates embedding such that an islet-poor region may be presented for sectioning. Overall, this method leads to low yield and clumped islet distribution in the resulting sections.
This new method is a simplified and improved approach for the preparation of islet sections. Islets are concentrated in a small volume and then placed on the smooth surface of a microscope slide to form a small disc, with the islets in a single plane. The Histogel-islet disc is subsequently processed for paraffin embedding in a shortened dehydration and xylene infiltration protocol. The previous approach, which concentrates the islets in the bottom of a microfuge tube, is also carried out as a comparison. This new technique improves the yield of islets per section, the distribution of islets in each section, and takes less time to transfer the islet blocks to cassettes. This technique is useful for islet biologists or other scientists studying small pieces of tissue wishing to maximize productive use of a low-abundance tissue by measuring multiple outcomes on a single sample in its native tissue architecture.

<table>
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf tube</td>
			<td>Norgen Bioteck Corp</td>
			<td>P/N 10113&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ranin Classic Starter Kit</td>
			<td>ShopRanin</td>
			<td>17008708</td>
			<td></td>
		</tr>
		<tr>
			<td>Ranin ClassicPipette PR-2</td>
			<td>ShopRanin</td>
			<td>17008648</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (1000 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (200 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (20 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (10 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-401</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Formalin solution</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Heatblock</td>
			<td>VWR</td>
			<td>949312</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoGel</td>
			<td>Thermo Scientific</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose blue beads- Affi-gel</td>
			<td>Bio-Rad</td>
			<td>153-7301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Life technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processing cassette</td>
			<td>Simport</td>
			<td>M492-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Wraps</td>
			<td>Leica-Surgipath</td>
			<td>3801090</td>
			<td></td>
		</tr>
		<tr>
			<td>Citadel 2000 tissue processor</td>
			<td>Thermo-Shandon LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Laboratories, INC</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Chemical</td>
			<td>X5SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>McCormick Scientific</td>
			<td>39503002</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo-Shandon LLC</td>
			<td>Finesse ME+</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin antibody</td>
			<td>DAKO</td>
			<td>A0564</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucagon antibody</td>
			<td>Sigma</td>
			<td>G2654</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex fluor 594 secondary antibodies</td>
			<td>Life technologies</td>
			<td>A11076</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex fluor 488 secondary antibodies</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
	</tbody>
</table>

pancreatic islet embedding for paraffin sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

An illustrated schematic of the steps to prepare the gel disc is shown in Figure 1. This gel disc method results in paraffin sections that contain a sufficient number of islets distributed in a single plane to allow meaningful quantification of outcomes. Figure 2 shows low-magnification images of the resulting sections to illustrate the number of islets captured per section. In general, &#62;35 islets were visible in each section...

Watch this Scientific Journal Video about Pancreatic Islet Embedding for Paraffin Sections at JoVE.com

Pancreatic Islet Embedding for Paraffin Sections

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

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