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In This Article

  • Summary
  • Abstract
  • Protocol
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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&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.

Abstract

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&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.

Protocol

1. Microtomy for Unstained Sections

  1. Set up water bath and microtome as for standard paraffin microtomy. Use positively charged slides and pre-label with case number, pancreas region (sample) and slide number. Place paraffin block in the microtome chuck with the cassette label on the left side.
  2. Follow normal microtomy procedures, section into block until tissue is uniformly encountered. Produce a ribbon of serial sections (4 ΞΌm thick, 3-6 depending on number of initial required stains (See Case Submission Form, Appendix 1)). Separate sections in the ribbon, pick up each in order, and place on slides with order numbered wi....

Discussion

Standardization of IHC procedures is critical for image analysis, particularly when using computer-based algorithms across large numbers of slides over time. The IHC staining procedure described in this report will allow batch analysis of a given donor's samples using an autostainer within an 8-hour work day and are modified from a previous report 5. Digitized whole slide images of each stained slide made available to nPOD-affiliated investigators through the web-based online pathology system. Investiga.......

Disclosures

No conflicts of interest declared.

Acknowledgements

The authors thank the donors' families and the Organ Procurement Organizations involved in this research and Emily Montgomery, Robert Pietras, Ann Fu, Mitali Agarwal, and Roshan Agarwal for their expert assistance. This work was funded by the Juvenile Diabetes Research Foundation (M.C-T.) in support of the Network for Pancreatic Organ Donors with Diabetes.

....

Materials

NameCompanyCatalog NumberComments
Name of the reagentCompanyCatalogue numberComments
30% Hydrogen Peroxide (H2O2)FisherScientificH325-500Endogenous peroxidase blocking
ABC-Alkaline Phosphatase (AP)VectorAK-5000AP conjugate
Antibody DiluentInvitrogen003218Primary antibody
Avidin blockerVectorSP-2001Block endogenous biotin
Biotinylated Goat anti-guinea pig VectorBA-7000Secondary antibody
Bluing ReagentFisherScientific7301Leica autostainer
Citrate buffer, pH 6.0BioGenexHK086-9KAntigen retrieval
Clarifier 1FisherScientific7401Leica autostainer
Cytoseal XYLFisherScientific8312-4Coverslip mountant
DABVectorSK-4100HRP chromogen
DEEBDAKOS2003Endogenous AP blocking reagent
Eosin Y AlcoholicFisherScientific71204Leica autostainer
Ethanols- 100%, 95%, 90%, 70%FisherScientificVariousDeparaffinization and coverslipping
HematoxylinDAKOS3301Dako autostainer
Hematoxylin 7211FisherScientific7211Leica autostainer
IgG (all host species for primary antibodies)VariousVariousNegative controls for primaries
Liquid Permanent Red (LPR)DAKOK0640AP chromogen
Mach2 APBiocare MedicalMALP521LGoat anti-Mouse AP conjugate
Mach2 HRPBiocare MedicalMHRP520LGoat anti-Mouse HRP conjugate
Mach2 HRPBiocare MedicalRHRP520LGoat anti-Mouse HRP conjugate
MethanolFisherScientificVariousH2O2 diluent
Normal goat serumVectorS-1000IHC blocking reagent
SniperBiocare MedicalBS966MIHC blocking reagent
TBST 20XThermoScientificTA-999-TTIHC buffer
Triology 20xCell Marque920P-06Antigen Retrieval
XyleneFisherScientificVariousDeparaffinization and coverslipping

Table 1. Specific reagents.

Primary AntibodyHost speciesVendorCat. #Comments and Antigen Retrieval
ParaffinΒ Β Β Β 
AmylaseMouseSanta CruzSC-46657Citrate
AmylinMouseSerotecMCA11267Citrate
Carbonic anhydrase 19.9MouseAbcamab15146Β 
Caspase-3, cleavedRabbitCell Signaling9961Citrate or Trilogy
CD20MouseDAKOM0755Citrate or Trilogy
CD3RabbitDAKOA0452Citrate
CD34MouseCell Signaling3569Citrate
CD4MouseDAKOM7310Citrate
CD45MouseDAKOM0754Citrate
CD68MouseDAKOM0876Citrate
CD8MouseDAKOM7103Citrate
Chromogranin ARabbitDAKOA0430Citrate
CK17MouseDAKOM7046Citrate
CK19MouseDAKOM0772Citrate
CK7MouseDAKOM7018Citrate
C-peptideRabbitCell Signaling4593Citrate
Foxp3Rate Bioscience14-4776-80Citrate
GhrelinGoatSanta CruzSC-10386Β 
GhrelinRabbitAbcamab85104Β 
GlucagonRabbitDAKOA0565Β 
GlucagonMouseAbcamab10988Citrate
GlucagonGuinea PigBachemT-5037 Citrate
Glut-1MouseAbcamab40084Trilogy
Glut-2 RabbitSanta CruzSC-9117Not expressed in human islets
InsulinGuinea PigDAKOA0564Β 
Ki-67MouseDAKOM7240Citrate
MafaRabbitNovus BiologicalNB400-137ATrilogy
Pancreatic polypeptideRabbitInvitrogen18-0043Citrate
Pdx-1Guinea PigAbcamab47308Citrate or Trilogy
ProinsulinMouseNovocastraNCL-proin-1G4Β 
ProinsulinMouseDevelopmental Studies Hybridoma BankGS-9A8Citrate
SecretagoginRabbitSigmaHPA 006641Citrate
Smooth muscle actinRabbitAbcamab5694Β 
SomatostatinRabbitDAKOA0566Β 
SynaptophysinMouseDAKOM0776Citrate
Fresh FrozenHost speciesVendorCat. #Comments
CD11bRatAbcamab6332Fix with 4% PF
CD11cRabbitSerotecAHP1226Β 
CD25 MouseNovus BiologicalNB 600-564Use overnight incubation for paraffin sections
CD94 MouseAbcamab61874Fix with acetone
GAD65MouseSanta CruzSC-130569Fix with 4% PF
HLA-ABCMouseDAKOM0736Fix with acetone or 4% PF

Table 2. Primary antibodies.

Name of the reagentCompanyCatalogue numberComments
Aperio Scanscope CSAperioΒ Digital slide scanner
DAKO Autostainer PlusDAKOΒ IHC stains
Label Matrix printing softwareBioCartaLM7UP57Slide label software
Leica XL AutostainerLeicaΒ H&E stains
Premium CoverglassFisherScientific12-548-5JCoverslip
Spectrum Information ManagerAperioΒ Scanner software
Superfrost Plus slidesFisherScientific12-550-15Positively charged slides
Vegetable steamerBlack and DeckerVariousAntigen retrieval
Zebra TLP 3742 CDWGTLP3742Slide label printer

Table 3. Specific supplies and equipment.

References

  1. In't Veld, P., Marichal, M. Microscopic anatomy of the human islet of Langerhans. Adv. Exp. Med. Biol. 654, 1-19 (2010).
  2. Matveyenko, A. V., Butler, P. C. Relationship between beta-cell mass and diabetes onset. ....

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Staining ProtocolsHuman Pancreatic IsletsIslet AreaEndocrine Cell CompositionProcessing And Staining ProcedureHistopathologyImmunolocalization ExaminationsStandardized ProceduresTransverse SectionsPancreatic HeadPancreas TailSerial SectioningHistopathological CharacterizationIslet Endocrine CellsReplicationT cell InfiltratesExocrine CompartmentDuctular Components

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