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 can be obtained, serial sections of the same islets can be analyzed, increasing the number of distinct outcomes that can be measured on the same experimental sample. Although optimized here for pancreatic islets, the technique could be applied to other low-abundance material, including those with small pieces, friable nature, or viscous samples that would otherwise be difficult to process. This method is optimized for fresh-fixed islets and has not been tested for other types of material, such as previously frozen islets. These sections may also be useful for in situ hybridization assays to detect DNA or RNA. The addition of extra gel serves two purposes: to maximize islet collection and to protect the islet-rich disc center from disruption during handling/transferring. The thin gel disc solidifies quickly and allows shortened dehydration and xylene infiltration steps for paraffin embedding. Forming the disc on a flat surface rather than in a secondary container (microfuge tube or culture plate well) makes it easier to physically transfer the disc to the cassette for processing. When compared with the disc technique results, the microtube technique had reduced islet yield and clumped islet distribution, although the non-planar tissue arrangement could generate a higher number of islet-containing sections.
The critical steps of the protocol include tissue fixation, formation of the gel disc, paraffin embedding, and sectioning. With respect to fixation, as tested by H&#38;E and immunofluorescence for insulin and glucagon, no difference was detected between PFA and formalin, or fixation duration ranging from 10 - 30 minutes (not shown). However, fixation intensity and duration should be optimized for end-user needs. The principal innovation contained in this protocol is the preparation of the gel islet disc; critical steps are summarized in Figure 1. Key steps include using a small volume of gel to concentrate tissue in a small area and forming the disc on a flat surface to arrange the tissue in a single plane for sectioning. Use of low-binding microfuge tubes and tips and a swinging bucket centrifuge for all spins improves tissue yield; islets stick to plastic after fixation. Addition of easily visible beads assists with disc formation, embedding and sectioning. Removing as much PBS as possible before adding the gel is critical to avoid diluting the agarose, which leads to poor solidification and difficulty transferring the disc to the paper. Keeping the thin disc intact while sliding it from the glass slide to the paper can be challenging. If a wrinkle occurs in the disc, allow it to return to the original position, add more gel and re-chill the slide. With respect to paraffin embedding, it is important that the disc be embedded flat-side down and parallel to the plane of cutting. A histotechnologist experienced with cutting paraffin sections is essential to the success of this procedure, since the material is concentrated at the leading edge of the block. Excessive facing off of the block may lead to loss of the sample. It is recommended to start collecting sections as soon as the blue beads are visible.
Fixation intensity and duration should be optimized for the end user&#39;s specific tissue and needs. The number of islets used per sample can be modified if necessary; however, reducing starting material generates sections with fewer islets. Thicker or larger discs can be generated using this technique; dehydration and xylene infiltration steps may need to be lengthened and should be optimized. If resulting sections contain only a partial disc shape, consider the possibility that the disc was not embedded parallel to the cutting surface. If too few sections are obtained, the technologist may have lost material during initial block preparation.
This method generates sections containing fixed islet tissue and, as such, can only be used for histological outcomes. To obtain a large number of islets per section, 200 - 250 IEQ starting material was used, which is challenging to generate for some experiment types. High quality sections are dependent on having an experienced histotechnologist.
The ability to generate 10+ sections containing many islets from a single experimental condition will allow quantification of multiple outcomes on the same material, improving experimental efficiency. Routine analysis of intact islets may lead to advances in understanding in tissue heterogeneity, not only on a cellular level, but also at the islet level, about which little is currently understood. Applying this technique to other limited-abundance tissue samples may offer benefits to other fields as well.

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 when 250 IEQ were used for the procedure, and &#62;10 sections (4 - 5 &#181;m) containing islets were obtained from each sample. Increasing the number of islets used for the procedure increased the number of islets in the sections. Islets in sections were structurally diverse and of varying sizes, supporting the concept that new and interesting data may be obtained using sections rather than techniques blind to inter-islet differences. The method worked equally well for rodent islets isolated in the laboratory (rat, mouse) and for human islets received after shipment by the IIDP. Rodent islets had noticeably more intact morphology after post-isolation recovery overnight in islet medium, with a smooth rounded surface and compact spherical shape (Figure 2A). Human islet morphology was similar when embedded on the day of arrival or when embedded after post-shipment recovery overnight, perhaps because the islets had already recovered from isolation stress prior to shipment. The microtube method yielded a smaller tissue cross-sectional area, with fewer islets per section, and densely packed islets and beads (Figure 2B). The microtube images shown in Figure 2B were the best we were able to obtain with this method; one of the samples had to be sent back to cut more sections because no islets were found in the first set of sections.
This technique generates high quality islet sections for histological and immunofluorescence staining (Figure 3). Insulin and glucagon staining showed the diverse composition and heterogeneity of islet architecture. The sections clearly recapitulated known architecture differences between human, mouse and rat islets, with human islets comprised of abundant &#945;-cells intermingled with beta cells, whereas mouse and rat islets showed similar architecture with a beta cell core and alpha cells in the periphery. Serial sections were obtainable; the panels in Figure 3 show serial sections of the same islet stained for H&#38;E (left) and immunofluorescence (right).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57931/57931fig1.jpg" /> 
Figure 1. Illustration of the critical steps for making the islet gel disc. Gel warmed to 70 &#176;C in a heat block (A) is transferred to the low binding microfuge tube containing the islet/bead pellet (B). The islets are resuspended in warm gel and transferred to a glass slide, spreading the material in a disc shape (C-D). Additional clean gel is transferred to the microfuge tube, then all remaining material is removed and spread circumferentially around the initial islet/bead-containing disc on the glass slide (E-F). After gelling on ice, the disc is transferred, flat side down, to histology paper (G), which is folded and placed in a cassette (H) for processing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57931/57931fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57931/57931fig2.jpg" /> 
Figure 2.&#160;Paraffin sections of gel discs contain a large number of well-distributed islets. (A) Low magnification panels (40X, 3 x 3 images stitched together, with 15% overlap) of mouse, rat or human islet sections stained with H&#38;E show that many islets are visible in each section. High magnification insets (200X) show representative islets. Left panels show sections of islets embedded immediately after isolation (mouse, rat) or shipment (human); right panels show islets embedded after overnight recovery in culture. Pale blue circles seen in 40X images are the beads included for visualization during embedding and sectioning. These particular samples are from a C57BL/6N mouse (1 year old) and BBDR rat (4 weeks old), islets isolated by ductal collagenase injection and Ficoll separation, and human T2D islets received from the IIDP. (B) 250 IEQ of human islets embedded per the old microfuge tube technique, for comparison. Low magnification panels (40X; a single unstitched image captured all material) and high mag insets (200X) show representative sections. Culture medium was RPMI with 10% FBS, penicillin/streptomycin, and 5.5 mmol/L glucose. Scale bars for low magnification panels are 1000 &#181;m; scale bars for high magnification panels are 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57931/57931fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57931/57931fig3.jpg" /> 
Figure 3. Representative histochemical and immunofluorescence staining on serial islet sections. Sections of mouse, rat, and human islets embedded after overnight culture were stained for H&#38;E and imaged by brightfield microscopy (200X; left). An adjacent section was stained for insulin (red), glucagon (green), mounted in DAPI-containing media (blue) and imaged using fluorescent microscopy (200X; right). Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57931/57931fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

Murine Pancreatic Islet Isolation

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...