Pancreatic Islet Embedding for Paraffin Sections

Summary

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.

Abstract

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 µm sections can be cut from the same islet block. Using this method, histological 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.

Introduction

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 architecture^1,2,3. The conventional strategy to study in vivo structure and endocrine cell composition of pancreatic islets is by sectioning pancreas tissue^4,5. 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 function^5,6.

Ex vivo islet experiments often are analyzed using molecular studies of whole islets or histological or molecular studies of dispersed islet cells grown in monolayer^5,6. 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 analyses^5,7. 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 sectioning^10,11. 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.

Protocol

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

1. 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.
2. 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 °C. Although this step is not required to obtain sections, the islets are more intact after a recovery period (see Figure 2).

2. Islet Fixation

1. 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.
2. 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.
3. Add 1 mL of PBS. Centrifuge tube in a swinging bucket centrifuge until the speed reaches 200 x g and then halt the spin. Remove the supernatant. Repeat PBS wash, for a total of two washes.
4. Add 500 µL 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.
5. Remove the fixative with a P200 tip.
6. Add 1 mL of PBS. Centrifuge tube until the speed reaches 200 x g and then halt the spin. Remove the supernatant. Repeat PBS wash, for a total of two washes.
7. Proceed immediately to the next step.

3. Preparation of Islet Disc (Figure 1)

1. On first use, melt the gel (e.g., Histogel) at 70 °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.
2. Warm the gel (approximately 100 µL per sample) at 70 °C by placing microfuge tube containing the gel aliquot in a heat block set to 70 °C.
3. Transfer agarose blue beads (10 µL 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 (>5x the number of islets) to allow optimal distribution of islets in sections.
4. 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 µL 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 µL.
5. 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 µL micropipette tip with scissors or a blade and add 10 µL of beads to each islet tube, using a clean tip for each sample. Do not mix (to avoid losing islets).
6. Centrifuge islets and beads (brief spin to 200 x g). Remove as much PBS as possible with an uncut 10 µL micropipette tip.
7. 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 µL 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.
8. Using the first micropipette, add 15 - 20 µL 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 <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.
9. Using a second micropipette, add 20 µL 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 µL 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.
10. Prepare each disc individually and keep careful track of sample order if placing multiple discs on each slide.
11. 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.
12. Label biopsy processing/embedding tissue cassettes with pencil, one per sample. Dry the back of the slide to avoid wetting the blue paper.
13. 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.
14. 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.
15. Submerge the cassette in PBS in a beaker. Process for paraffin embedding the same day for optimal morphology.

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.

1. Immerse cassettes in 85% ethanol for 15 minutes.
2. Immerse cassettes in 95% ethanol for 15 minutes.
3. Immerse cassettes in 100% ethanol for 15 minutes. Repeat twice for a total of three washes.
4. Immerse cassettes in xylene for 15 minutes. Repeat twice for a total of three washes.
5. Immerse cassettes in molten paraffin for 10 minutes.
6. Transfer cassettes to fresh molten paraffin for 10 - 30 minutes.
7. 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.

5. Paraffin Sectioning and Staining

1. Label slides sequentially if serial sections are required.
2. Have 5 µm 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.
3. Store sections at room temperature. Sections are amenable to routine histological stains (e.g., H&E) and immunofluorescence (e.g., insulin, glucagon and DAPI). Optimize fixation for other antigens, if necessary.

Results

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, >35 islets were visible in each section...

Discussion

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' needs. Since multiple sections ...

Materials

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