Name: pancreatic islet embedding for paraffin sections
Uploaded: 2018-06-29T14:30:00.000+00:00
Duration: 9 min 9 s
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 isolated by collagenase ductal insufflation and Ficoll separation12 (rodent) or post-shipment islets from the Integrated Islet Distribution Program (IIDP13; human). Islets were handpicked with a P200 micropipette.</li>
	<li>To optimize islet morphology, allow islets to recover overnight in 10 mL complete islet medium (RPMI with 10% FBS, penicillin/streptomycin, and 5.5 mmol/L glucose) in a humidified chamber containing 5% CO2 at 37 &#176;C. Although this step is not required to obtain sections, the islets are more intact after a recovery period (see Figure 2).</li>
</ol>
2. Islet Fixation
<ol>
	<li>Using a low-binding P200 tip, handpick approximately 250 islet equivalents (IEQ)13 using a calibrated grid under a stereomicroscope into a 1.5 mL low binding microfuge tube. Low-binding tips and tubes reduce islet loss.</li>
	<li>Allow islets to settle to the bottom of the microfuge tube. Remove most supernatant with a P200 pipet tip, taking care not to remove any islets.</li>
	<li>Add 1 mL of PBS. Centrifuge tube in a swinging bucket centrifuge&#160;until the speed reaches 200 x g and then halt the spin. Remove the supernatant. Repeat PBS wash, for a total of two washes.</li>
	<li>Add 500 &#181;L of 10% formalin solution or 4% freshly made paraformaldehyde. Fix at room temperature for 30 minutes. For the outcomes tested in this method, these fixation methods were indistinguishable. Shorter fixation may also be possible; it is recommended to optimize fixation duration for desired outcome.</li>
	<li>Remove the fixative with a P200 tip.</li>
	<li>Add 1 mL of PBS. Centrifuge tube&#160;until the speed reaches 200 x g and then halt the spin. Remove the supernatant. Repeat PBS wash, for a total of two washes.</li>
	<li>Proceed immediately to the next step.</li>
</ol>
3. Preparation of Islet Disc (Figure 1)
<ol>
	<li>On first use, melt the gel (e.g., Histogel) at 70 &#176;C and make aliquots in 1.5 mL microfuge tubes for long term storage. Aliquot volume is not critical, since aliquots can be re-used.</li>
	<li>Warm the gel (approximately 100 &#181;L per sample) at 70 &#176;C by placing microfuge tube containing the gel aliquot in a heat block set to 70 &#176;C.</li>
	<li>Transfer agarose blue beads (10 &#181;L for each sample) into a 1.5 mL clean microfuge tube. Thoroughly resuspend the beads before pipetting. 
	NOTE: Blue beads are mixed with the islets to assist in visual identification of the embedded material in the agarose button and the paraffin block. The number of blue beads used is not critical to the outcome, but avoid excessive beads (&#62;5x the number of islets) to allow optimal distribution of islets in sections.</li>
	<li>Wash the blue beads with 1mL PBS, twice. For each wash, spin the beads 1 min at 800 x g. After the second wash, resuspend the beads in (n+2) x 10 &#181;L PBS, where n is the number of sample tubes; for example, for 2 sample tubes, the PBS volume to add to the beads would be 40 &#181;L.</li>
	<li>Centrifuge the microfuge tube containing islets (brief spin to 200 x g) and remove most of the supernatant. Cut off the tip of a 10 &#181;L micropipette tip with scissors or a blade and add 10 &#181;L of beads to each islet tube, using a clean tip for each sample. Do not mix (to avoid losing islets).</li>
	<li>Centrifuge islets and beads (brief spin to 200 x g). Remove as much PBS as possible with an uncut 10 &#181;L micropipette tip.</li>
	<li>Label the microscope slides for sample identification. Two to three samples can be prepared on each slide. Place the slide on the bench near the heat block with warmed gel. Cut the tips of three to five 20 &#181;L low binding micropipette tips per sample with a razor blade or scissors. Load two P20 micropipettes with cut tips to allow rapid switching from one to the other.</li>
	<li>Using the first micropipette, add 15 - 20 &#181;L of warm gel to the islets/beads microfuge tube; immediately mix, avoiding bubbles; and apply the liquid agar/islets/beads mixture to the slide to form a &#60;1 cm diameter disc. Place the disc near the edge of the slide, leaving space for the outer gel ring (next step). Tap the slide gently a few times to settle the islets and beads.</li>
	<li>Using a second micropipette, add 20 &#181;L of warm gel to the sample tube. Using the first micropipette, mix new gel with any remaining islets/beads, re-warming in the heat block if it starts to solidify, then apply this mixture to the slide, surrounding the original disc. Repeat as necessary, using additional pre-cut tips, until the disc is the desired size and thickness. 
	NOTE: Handling the gelled disc is easier if the outside ring is slightly thicker. Usually, 3 x 20 &#181;L of gel is sufficient. This step minimizes loss of islets by tube washes and adds islet-poor agarose to the outside of the disc to facilitate disc handling.</li>
	<li>Prepare each disc individually and keep careful track of sample order if placing multiple discs on each slide.</li>
	<li>Place the slides on a flat surface of wet ice, with a cover, for 10 minutes or until the gel solidifies. It is more difficult to slide the disc off the glass if it dries out.</li>
	<li>Label biopsy processing/embedding tissue cassettes with pencil, one per sample. Dry the back of the slide to avoid wetting the blue paper.</li>
	<li>Using the blunt edge of a razor blade, gently push the disc in each direction to free it from the glass. When it slides easily, slowly push the disc from the microscope slide onto the blue tissue paper. Place the disc, flat side down, directly on the paper.</li>
	<li>Keeping the disc flat, fold the paper around the disc to prevent movement, place the disc in folded paper in the cassette, and close the cassette. If the disc does not slide cleanly off the glass, add more gel and/or return the slide to ice for a few more minutes.</li>
	<li>Submerge the cassette in PBS in a beaker. Process for paraffin embedding the same day for optimal morphology.</li>
</ol>
4. Paraffin Embedding
NOTE: Process the islet gel discs to paraffin blocks using an abbreviated dehydration series as described below. This technique was optimized using an automated processor, but manual processing should produce similar results.
<ol>
	<li>Immerse cassettes in 85% ethanol for 15 minutes.</li>
	<li>Immerse cassettes in 95% ethanol for 15 minutes.</li>
	<li>Immerse cassettes in 100% ethanol for 15 minutes. Repeat twice for a total of three washes.</li>
	<li>Immerse cassettes in xylene for 15 minutes. Repeat twice for a total of three washes.</li>
	<li>Immerse cassettes in molten paraffin for 10 minutes.</li>
	<li>Transfer cassettes to fresh molten paraffin for 10 - 30 minutes.</li>
	<li>Carefully open the cassette and unwrap the blue paper. Remove the gel disc, keeping track of the flat surface that was opposed to the paper. Embed the disc in a small mold, with the flat (islet-containing) surface down, parallel to the cutting surface. For the plug, carefully remove it from the cassette and embed it in a small mold with the tip pointing towards the cutting surface.</li>
</ol>
5. Paraffin Sectioning and Staining
<ol>
	<li>Label slides sequentially if serial sections are required.</li>
	<li>Have 5 &#181;m paraffin sections cut by an experienced histotechnologist. To maximize yield, save all sections containing material. Generally, collecting 20 sections allows capture of most of the material.</li>
	<li>Store sections at room temperature. Sections are amenable to routine histological stains (e.g., H&#38;E) and immunofluorescence (e.g., insulin, glucagon and DAPI). Optimize fixation for other antigens, if necessary.</li>
</ol>

<ol>
	<li>Kim, A., Miller, K., Jo, J., Kilimnik, G., Wojcik, P., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+architecture:+A+comparative+study.">Islet architecture: A comparative study.</a> Islets. 1 (2), 129-136 (2009).</li><li>Steiner, D. J., Kim, A., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+plasticity:+Interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity: Interspecies comparison of islet architecture and composition.</a> Islets. 2 (3), 135-145 (2010).</li><li>Campbell-Thompson, M. 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. 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=Insulin+demand+regulates+&#946;+cell+number+via+the+unfolded+protein+response.">Insulin demand regulates &#946; cell number via the unfolded protein response.</a> The Journal of Clinical Investigation. 125 (10), 3831-3846 (2015).</li><li>Stamateris, R. E., 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=Glucose+Induces+Mouse+&#946;-Cell+Proliferation+via+IRS2,+MTOR,+and+Cyclin+D2+but+Not+the+Insulin+Receptor.">Glucose Induces Mouse &#946;-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor.</a> Diabetes. 65 (4), 981-995 (2016).</li><li>Blodgett, D. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+HistoGelTM-embedded+specimens+for+use+in+veterinary+diagnostic+pathology.">Evaluation of HistoGelTM-embedded specimens for use in veterinary diagnostic pathology.</a> Journal of Veterinary Diagnostic Investigation: Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 24 (4), 710-715 (2012).</li><li>Cozar-Castellano, I., Takane, K. K., Bottino, R., Balamurugan, A. N., Stewart, A. 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. 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=Glucose+infusion+in+mice:+A+new+model+to+induce+beta-cell+replication.">Glucose infusion in mice: A new model to induce beta-cell replication.</a> Diabetes. 56 (7), 1792-1801 (2007).</li></ol>

The authors have no conflicts of interest to declare.

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&amp;nbsp;</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 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-430</td><td></td></tr><tr><td>Low Binding Tip (200 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-412</td><td></td></tr><tr><td>Low Binding Tip (20 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-404</td><td></td></tr><tr><td>Low Binding Tip (10 &amp;mu;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&#039;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 ...

pancreatic-islet-embedding-for-paraffin-sections

Research

JoVE Journal

Biology

12.7K Views.  UMass Medical School. 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.

Video: Pancreatic Islet Embedding for Paraffin Sections

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Medicine

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

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.

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 ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Developmental Biology

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Bioengineering

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

Neuroscience

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

Immunology and Infection

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Slicing Human Pancreas for Islet Function Research

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...

Pancreatic Islet Embedding for Paraffin Sections

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Collection Protocol for Human Pancreas

Staining Protocols for Human Pancreatic Islets

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

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology