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. <....

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 D....

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

List of materials used in this article

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