Department of Pathology, Immunology, and Laboratory Medicine, University of Florida
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Campbell-Thompson, M. L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. Staining Protocols for Human Pancreatic Islets. J. Vis. Exp. (63), e4068, doi:10.3791/4068 (2012).
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.
1. Microtomy for Unstained Sections
2. H&E Staining
3. IHC Set-up
4. IHC Deparaffinization and Antigen Retrieval
5. First Primary Antibodies (Ki-67, CD3, Pancreatic Polypeptide)
6. Second Primary Antibodies (Insulin, Glucagon)
8. Slide Labeling
9. Slide Scanning
10. Representative Results
These staining procedures were developed for the JDRF Network for Pancreatic Organ donors with Diabetes (nPOD) to provide baseline characterization of paraffin and fresh frozen blocks. Each donor has a baseline characterization performed comprised of staining 2 blocks from each pancreas region (head, body, and tail) and the spleen (Figure 1, see also case submission form, Appendix 1). The H&E staining is conducted using an autostainer programmed as for a clinical facility with clinical-grade solutions used throughout. As additional donor blocks are sectioned, each has a slide taken for H&E to show tissue morphology. When blocks are sectioned again, as for distribution to investigators, deeper levels will also have slides taken for representative H&E slides to document the entire block's tissue morphology as well as numbering serial sections at each level. Stained slides are labeled with case identification, pancreas region, block number, stain and date (Figure 2) and scanned into the online pathology information management system. Representative images from a control donor are shown in Figure 3 at both low and high magnifications to show expected results following this procedure. The slide stained with pancreatic polypeptide (D,H) was assayed on a different day with the assay performed manually and shows reduced staining intensity of the hematoxylin counterstain. Differences in staining intensity of primary antibodies or counterstain between assays is to be avoided particularly if using image analysis otherwise slides with require individual color correction to achieve the same cell areas or counts.
Each laboratory should expect to optimize antibody concentrations as lot-to-lot variation and other reagents and conditions can impact staining intensity and specificity. With acquisition of new reagents, staining procedures are validated with known positive and negative control samples to achieve the same degree of stain intensity as with former reagents. Additional antibodies optimized in the nPOD pathology core are provided in Table 2 as a reference source and have been used for multilabeling immunofluorescence assays for confocal microscopy.
Figure 1. Dako staining grid. This schematic depicts a routine analysis from a nPOD donor pancreas performed on a Dako autostainer. The nPOD donors are coded with a 4-digit identification number. Two slide trays are shown with slides organized by stain. Alternate slide configurations are expected depending on numbers of donor slides. Positive controls include inclusion of a known positive donor for the two double stains and donor spleen for Ki-67 and CD3. Negative controls are twofold and include two slides from one donor pancreas block incubated with immunoglobulin (Ig) from the host species of the primary antibodies and two slides from donor spleen for insulin and glucagon.
Figure 2. Representative double stained slide. A typical finished slide is shown with slide labeling parameters and tissue placement before digital scanning is performed.
Figure 3. Representative H&E and IHC stains in pancreatic sections. Sections from the pancreas of an adult female organ donor (6096-04 PanHead) were stained and digital scans performed as described in the protocol. Images were obtained using the ImageScope viewing program of the entire section (A-D) and from one islet (E-H). The expected islet stain intensities for insulin, glucagon, and pancreatic polypeptide are observed for sections B-D as section D delineates that this pancreas head block contains a small portion of the uncinate region or ventral pancreatic lobe as all islets contain mainly pancreatic polypeptide-positive cells. Note that the hematoxylin counterstain in panel D is too light while the hematoxylin counterstain intensities in B and C are optimal. Panels E-H show an islet from the dorsal lobe with the expected distribution of β-cells and α-cells. A few pancreatic polypeptide cells are found in the lower left portion of the islet (H). A, E- H&E; B, F- Ki67+Insulin; C, G- CD3+Glucagon; D, H- Pancreatic polypeptide. A-D, 0.2x, E-H- 12.8x.
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. Investigators can review donor demographics, laboratory data, and other pertinent clinical history along with the slides and are able to choose blocks most suitable for their studies (see http://www.jdrfnpod.org/online-pathology.php for more information). Such an online pathology system serves as a "virtual biobank". A further improvement to this system will be implemented whereby slide labels incorporate a bar code to track serial sections and levels for each block with an eventual goal of 3-D reconstruction of the pancreas.
The two double staining procedures were primarily chosen to determine islet β-cell composition (insulin) in concert with cellular replication (Ki-67) given the importance of understanding mechanisms for adult β-cell regeneration in type 1 diabetes 5,6. As the second double IHC assay is performed on serial sections to the first, each islet is characterized for both β-cell (insulin) and α-cell composition (glucagon). In addition, the presence of T-lymphocytes (CD3) is determined in conjunction with α-cell staining for two main reasons. The basis of T-cell mediated autoimmunity in progression of type 1 diabetes has been clearly appreciated yet the nature of the initiating agents (viral, bacterial, inflammatory cells) or the composition of the chronic infiltrate with resultant β-cell dysfunction and demise are yet to be defined (reviewed in 7,8). Second, donors with type 1 diabetes have a well characterized deficiency of β-cells so that combination of the CD3 and glucagon stains ensures identification of islets even when β-cell loss is severe9.
No conflicts of interest declared.
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.
|30% Hydrogen Peroxide (H2O2)||Fisher Scientific||H325-500||Endogenous peroxidase blocking|
|ABC-Alkaline Phosphatase (AP)||Vector Laboratories||AK-5000||AP conjugate|
|Antibody Diluent||Invitrogen||003218||Primary antibody|
|Avidin blocker||Vector Laboratories||SP-2001||Block endogenous biotin|
|Biotinylated Goat anti-guinea pig||Vector Laboratories||BA-7000||Secondary antibody|
|Bluing Reagent||Fisher Scientific||7301||Leica autostainer|
|Citrate buffer, pH 6.0||BioGenex||HK086-9K||Antigen retrieval|
|Clarifier 1||Fisher Scientific||7401||Leica autostainer|
|Cytoseal XYL||Fisher Scientific||8312-4||Coverslip mountant|
|DAB||Vector Laboratories||SK-4100||HRP chromogen|
|DEEB||Dako||S2003||Endogenous AP blocking reagent|
|Eosin Y Alcoholic||Fisher Scientific||71204||Leica autostainer|
|Ethanols- 100%, 95%, 90%, 70%||Fisher Scientific||Various||Deparaffinization and coverslipping|
|Hematoxylin 7211||Fisher Scientific||7211||Leica autostainer|
|IgG (all host species for primary antibodies)||Various||Various||Negative controls for primaries|
|Liquid Permanent Red (LPR)||Dako||K0640||AP chromogen|
|Mach2 AP||Biocare Medical||MALP521L||Goat anti-Mouse AP conjugate|
|Mach2 HRP||Biocare Medical||MHRP520L||Goat anti-Mouse HRP conjugate|
|Mach2 HRP||Biocare Medical||RHRP520L||Goat anti-Mouse HRP conjugate|
|Methanol||Fisher Scientific||Various||H2O2 diluent|
|Normal goat serum||Vector Laboratories||S-1000||IHC blocking reagent|
|Sniper||Biocare Medical||BS966M||IHC blocking reagent|
|TBST 20X||Thermo Fisher Scientific, Inc.||TA-999-TT||IHC buffer|
|Triology 20x||Cell Marque||920P-06||Antigen Retrieval|
|Xylene||Fisher Scientific||Various||Deparaffinization and coverslipping|
|Table 1. Specific reagents.|
|Amylase||Santa Cruz Biotechnology, Inc.||SC-46657||Mouse|
|Carbonic anhydrase 19.9||Abcam||ab15146||Mouse|
|Caspase-3, cleaved||Cell Signaling Technology||9961||Rabbit|
|CD34||Cell Signaling Technology||3569||Mouse|
|C-peptide||Cell Signaling Technology||4593||Rabbit|
|Ghrelin||Santa Cruz Biotechnology, Inc.||SC-10386||Goat|
|Glut-2||Santa Cruz Biotechnology, Inc.||SC-9117||Rabbit|
|Proinsulin||Developmental Studies Hybridoma Bank||GS-9A8||Mouse|
|Smooth muscle actin||Abcam||ab5694||Rabbit|
|CD25||Novus Biological||NB 600-564||Mouse|
|GAD65||Santa Cruz Biotechnology, Inc.||SC-130569||Mouse|
|Table 2. Primary antibodies.|
|Aperio Scanscope CS||Aperio Technologies||Digital slide scanner|
|DAKO Autostainer Plus||Dako||IHC stains|
|Label Matrix printing software||BioCarta||LM7UP57||Slide label software|
|Leica XL Autostainer||Leica Microsystems||H&E stains|
|Premium Coverglass||Fisher Scientific||12-548-5J||Coverslip|
|Spectrum Information Manager||Aperio Technologies||Scanner software|
|Superfrost Plus slides||Fisher Scientific||12-550-15||Positively charged slides|
|Vegetable steamer||Black and Decker||Various||Antigen retrieval|
|Zebra TLP 3742||CDWG||TLP3742||Slide label printer|
|Table 3. Specific supplies and equipment.|