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.

1. Microtomy for Unstained Sections
<ol>
 <li>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. </li>
 <li>Follow normal microtomy procedures, section into block until tissue is uniformly encountered. Produce a ribbon of serial sections (4 &mu;m 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 with pencil. Denote if a section is unused by numbering sections in exact order. Maintain the same orientation on the slide as found in the cassette with the slide label oriented to the left. Dry overnight at room temperature then proceed with staining or distribution to investigators.</li>
 <li>Reseal surface of paraffin block with thin layer of paraffin to preserve tissue and archive at room temperature or -20 &deg;C.</li>
 <li>For subsequent microtomy, maintain numbering for serial sections at each level as above. Obtain 1 additional slide and stain by H&amp;E slide for each ~100 &mu;M level within each block.</li>
</ol>
2. H&amp;E Staining
<ol>
 <li>Place the first serial sectioned slide from each block in a slide rack then in the first tray on the autostainer. </li>
 <li>Select the standard H&amp;E stain program and proceed as usual. </li>
 <li>When finished staining, remove the slides from autostainer and place in hood for coverslipping. </li>
 <li>Coverslip using standard technique. Dry thoroughly and label with a printed label (see section 12). </li>
</ol>
3. IHC Set-up
<ol>
 <li>Select the Dako program for double stains and input numbers of slides. Prepare TBST and citrate buffers, antibodies, and other reagents according to specified volumes (Table 1). Load the reagent tray and slides. Rotation of clean and waste lines are programmed according to manufacturer's recommendations. </li>
 <li>If performing these assays manually, prepare estimated volumes. Incubate slides in a humidified chamber to avoid slides drying out at any step. Follow the same procedure for each reagents as when used for the autostainer. </li>
</ol>
4. IHC Deparaffinization and Antigen Retrieval
<ol>
 <li>Put slides in xylene for 5 minutes. Repeat once. Do not allow the slides to dry out at any subsequent point until indicated as this will result in excessive background and poor staining.</li>
 <li>Transfer slides to 100% ethanol for 2 minutes. Repeat once.</li>
 <li>Put slides in freshly prepared 3% H2O2 in methanol for 10 minutes. </li>
 <li>Dip slides in 90% Ethanol and then place in 90% Ethanol for 3 minutes.</li>
 <li>Transfer slides to 70% Ethanol for 1 minute.</li>
 <li>Rinse slides in de-ionized water.</li>
 <li>Preheat steamer and citrate buffer in steamer for 5 minutes. </li>
 <li>Add slides to citrate buffer in the steamer and steam for 30 minutes.</li>
 <li>Immediately transfer slides to water and hold until slides cool to room temperature (~20 minutes).</li>
 <li>Transfer slides to the Dako autostainer racks according to stain sequence (Figure 1) and run the double staining program. The major steps of the autostainer program are listed so that manual runs can duplicate. </li>
</ol>
5. First Primary Antibodies (Ki-67, CD3, Pancreatic Polypeptide)
<ol>
 <li>Block slides with Sniper solution for 15 minutes. Wash twice with TBST.</li>
 <li>Incubate slides in primary antibody for 30 minutes. Wash twice with TBST.</li>
 <li>Incubate with Mach2 goat anti-mouse (Ki-67) or anti-rabbit (CD3, pancreatic polypeptide) horse radish peroxidase (HRP) polymer for 30 minutes. Wash twice with TBST.</li>
 <li>Apply 3,3 -diaminobenzidine tetrahydrochloride (DAB) to slides for 4 minutes. Wash twice with water.</li>
 <li>Slides incubated with pancreatic polypeptide are held on the Dako when being assayed concurrently and TBST used for all subsequent steps while the remaining slides are incubated with the second set of primary antibodies. Alternatively, for manual assays, proceed to section 6.</li>
</ol>
6. Second Primary Antibodies (Insulin, Glucagon)
<ol>
 <li>Incubate slides in DEEB for 20 minutes. Wash twice with TBST.</li>
 <li>Block with 10% normal goat serum in 20% avidin blocker in TBST (insulin) or Sniper (glucagon) in TBST for 20 minutes. Wash twice with TBST.</li>
 <li>Incubate with insulin or glucagon primary antibody for 15 minutes. Wash twice with TBST.</li>
 <li>Insulin: Incubate slides in biotinylated goat anti-guinea pig at 1:300 dilution for 30 minutes. Wash twice with TBST. Incubate with standard vector ABC-AP reagent for 30 minutes. Wash twice with TBST.</li>
 <li>Glucagon: Incubate slides in buffer during for 30 minutes followed by goat anti-mouse alkaline phosphatase (AP) polymer for 30 minutes. Wash twice with TBST.</li>
 <li> While slides are incubated in conjugates, warm the LPR buffer to room temperature. Add 1 drop of liquid permanent red (LPR) per 3 mL buffer immediate before use and place in Dako reagent rack. Incubate slides in LPC for 4 minutes. Wash twice with water.</li>
 <li>Incubate slides in hematoxylin for 1 minute. Rinse twice with water.</li>
 <li>Incubate slides in TBS pH 7.6 for 1 minute. Rinse twice with water.</li>
 <li>Unload slides from DAKO Autostainer.</li>
 <li>Immediately dehydrate slides stained for pancreatic polypeptide. For all double stained slides, dry for at least 1 hour then dehydrate.</li>
</ol>
7. Dehydration
<ol>
 <li>Place slides in 80% ethanol for 1 minute.</li>
 <li>Dip slides in 95% ethanol.</li>
 <li>Hold slides in 95% ethanol for 30 seconds.</li>
 <li>Transfer slides to 100% ethanol and hold 1 minute. Repeat.</li>
 <li>Dip slides in xylene.</li>
 <li>Hold slides in xylene for 1 minute. Repeat.</li>
 <li>Immediately mount coverslips on slides using cytoseal.</li>
</ol>
8. Slide Labeling
<ol>
 <li>Enter the case identification information including organ and block number, stain, and date and print. Serial section number is optional for the first slide stains of each block. Subsequent levels are denoted by numbering.</li>
 <li>Place labels on slides.</li>
</ol>
9. Slide Scanning
<ol>
 <li>Place stained slides in scanner tray and scan according to instrumentation. </li>
 <li>Organize slides by donor and tissue type (pancreas head, body, tail, spleen).</li>
 <li>Archive stained slides at room temperature and any remaining unstained slides at -20 &deg;C until used. </li>
</ol>
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&amp;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&amp;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&amp;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.
<img src="/files/ftp_upload/4068/4068fig1.jpg" alt="Figure 1"/> 
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. 
<img src="/files/ftp_upload/4068/4068fig2.jpg" alt="Figure 2"/> 
Figure 2. Representative double stained slide. A typical finished slide is shown with slide labeling parameters and tissue placement before digital scanning is performed.
<img src="/files/ftp_upload/4068/4068fig3.jpg" alt="Figure 3"/> 
Figure 3. Representative H&amp;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 &beta;-cells and &alpha;-cells. A few pancreatic polypeptide cells are found in the lower left portion of the islet (H). A, E- H&amp;E; B, F- Ki67+Insulin; C, G- CD3+Glucagon; D, H- Pancreatic polypeptide. A-D, 0.2x, E-H- 12.8x.

<ol>
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No conflicts of interest declared.

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...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>30% Hydrogen Peroxide (H2O2)</td>
 <td>FisherScientific</td>
 <td>H325-500</td>
 <td>Endogenous peroxidase blocking</td>
 </tr>
 <tr>
 <td>ABC-Alkaline Phosphatase (AP)</td>
 <td>Vector</td>
 <td>AK-5000</td>
 <td>AP conjugate</td>
 </tr>
 <tr>
 <td>Antibody Diluent</td>
 <td>Invitrogen</td>
 <td>003218</td>
 <td>Primary antibody</td>
 </tr>
 <tr>
 <td>Avidin blocker</td>
 <td>Vector</td>
 <td>SP-2001</td>
 <td>Block endogenous biotin</td>
 </tr>
 <tr>
 <td>Biotinylated Goat anti-guinea pig </td>
 <td>Vector</td>
 <td>BA-7000</td>
 <td>Secondary antibody</td>
 </tr>
 <tr>
 <td>Bluing Reagent</td>
 <td>FisherScientific</td>
 <td>7301</td>
 <td>Leica autostainer</td>
 </tr>
 <tr>
 <td>Citrate buffer, pH 6.0</td>
 <td>BioGenex</td>
 <td>HK086-9K</td>
 <td>Antigen retrieval</td>
 </tr>
 <tr>
 <td>Clarifier 1</td>
 <td>FisherScientific</td>
 <td>7401</td>
 <td>Leica autostainer</td>
 </tr>
 <tr>
 <td>Cytoseal XYL</td>
 <td>FisherScientific</td>
 <td>8312-4</td>
 <td>Coverslip mountant</td>
 </tr>
 <tr>
 <td>DAB</td>
 <td>Vector</td>
 <td>SK-4100</td>
 <td>HRP chromogen</td>
 </tr>
 <tr>
 <td>DEEB</td>
 <td>DAKO</td>
 <td>S2003</td>
 <td>Endogenous AP blocking reagent</td>
 </tr>
 <tr>
 <td>Eosin Y Alcoholic</td>
 <td>FisherScientific</td>
 <td>71204</td>
 <td>Leica autostainer</td>
 </tr>
 <tr>
 <td>Ethanols- 100%, 95%, 90%, 70%</td>
 <td>FisherScientific</td>
 <td>Various</td>
 <td>Deparaffinization and coverslipping </td>
 </tr>
 <tr>
 <td>Hematoxylin</td>
 <td>DAKO</td>
 <td>S3301</td>
 <td>Dako autostainer</td>
 </tr>
 <tr>
 <td>Hematoxylin 7211</td>
 <td>FisherScientific</td>
 <td>7211</td>
 <td>Leica autostainer</td>
 </tr>
 <tr>
 <td>IgG (all host species for primary antibodies)</td>
 <td>Various</td>
 <td>Various</td>
 <td>Negative controls for primaries</td>
 </tr>
 <tr>
 <td>Liquid Permanent Red (LPR)</td>
 <td>DAKO</td>
 <td>K0640</td>
 <td>AP chromogen</td>
 </tr>
 <tr>
 <td>Mach2 AP</td>
 <td>Biocare Medical</td>
 <td>MALP521L</td>
 <td>Goat anti-Mouse AP conjugate</td>
 </tr>
 <tr>
 <td>Mach2 HRP</td>
 <td>Biocare Medical</td>
 <td>MHRP520L</td>
 <td>Goat anti-Mouse HRP conjugate </td>
 </tr>
 <tr>
 <td>Mach2 HRP</td>
 <td>Biocare Medical</td>
 <td>RHRP520L</td>
 <td>Goat anti-Mouse HRP conjugate</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>FisherScientific</td>
 <td>Various</td>
 <td>H2O2 diluent</td>
 </tr>
 <tr>
 <td>Normal goat serum</td>
 <td>Vector</td>
 <td>S-1000</td>
 <td>IHC blocking reagent</td>
 </tr>
 <tr>
 <td>Sniper</td>
 <td>Biocare Medical</td>
 <td>BS966M</td>
 <td>IHC blocking reagent</td>
 </tr>
 <tr>
 <td>TBST 20X</td>
 <td>ThermoScientific</td>
 <td>TA-999-TT</td>
 <td>IHC buffer</td>
 </tr>
 <tr>
 <td>Triology 20x</td>
 <td>Cell Marque</td>
 <td>920P-06</td>
 <td>Antigen Retrieval</td>
 </tr>
 <tr>
 <td>Xylene</td>
 <td>FisherScientific</td>
 <td>Various</td>
 <td>Deparaffinization and coverslipping </td>
 </tr>
 </tbody> 
</table>
Table 1. Specific reagents.
<table border="1">
 <tbody>
 <tr>
 <td>Primary Antibody</td>
 <td>Host species</td>
 <td>Vendor</td>
 <td>Cat. #</td>
 <td>Comments and Antigen Retrieval</td>
 </tr>
 <tr>
 <td>Paraffin</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Amylase</td>
 <td>Mouse</td>
 <td>Santa Cruz</td>
 <td>SC-46657</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Amylin</td>
 <td>Mouse</td>
 <td>Serotec</td>
 <td>MCA11267</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Carbonic anhydrase 19.9</td>
 <td>Mouse</td>
 <td>Abcam</td>
 <td>ab15146</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Caspase-3, cleaved</td>
 <td>Rabbit</td>
 <td>Cell Signaling</td>
 <td>9961</td>
 <td>Citrate or Trilogy </td>
 </tr>
 <tr>
 <td>CD20</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M0755</td>
 <td>Citrate or Trilogy </td>
 </tr>
 <tr>
 <td>CD3</td>
 <td>Rabbit</td>
 <td>DAKO</td>
 <td>A0452</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CD34</td>
 <td>Mouse</td>
 <td>Cell Signaling</td>
 <td>3569</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CD4</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M7310</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CD45</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M0754</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CD68</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M0876</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CD8</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M7103</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Chromogranin A</td>
 <td>Rabbit</td>
 <td>DAKO</td>
 <td>A0430</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CK17</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M7046</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CK19</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M0772</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>CK7</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M7018</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>C-peptide</td>
 <td>Rabbit</td>
 <td>Cell Signaling</td>
 <td>4593</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Foxp3</td>
 <td>Rat</td>
 <td>e Bioscience</td>
 <td>14-4776-80</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Ghrelin</td>
 <td>Goat</td>
 <td>Santa Cruz</td>
 <td>SC-10386</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ghrelin</td>
 <td>Rabbit</td>
 <td>Abcam</td>
 <td>ab85104</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glucagon</td>
 <td>Rabbit</td>
 <td>DAKO</td>
 <td>A0565</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glucagon</td>
 <td>Mouse</td>
 <td>Abcam</td>
 <td>ab10988</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Glucagon</td>
 <td>Guinea Pig</td>
 <td>Bachem</td>
 <td>T-5037 </td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Glut-1</td>
 <td>Mouse</td>
 <td>Abcam</td>
 <td>ab40084</td>
 <td>Trilogy</td>
 </tr>
 <tr>
 <td>Glut-2 </td>
 <td>Rabbit</td>
 <td>Santa Cruz</td>
 <td>SC-9117</td>
 <td>Not expressed in human islets</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Guinea Pig</td>
 <td>DAKO</td>
 <td>A0564</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ki-67</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M7240</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Mafa</td>
 <td>Rabbit</td>
 <td>Novus Biological</td>
 <td>NB400-137A</td>
 <td>Trilogy</td>
 </tr>
 <tr>
 <td>Pancreatic polypeptide</td>
 <td>Rabbit</td>
 <td>Invitrogen</td>
 <td>18-0043</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Pdx-1</td>
 <td>Guinea Pig</td>
 <td>Abcam</td>
 <td>ab47308</td>
 <td>Citrate or Trilogy</td>
 </tr>
 <tr>
 <td>Proinsulin</td>
 <td>Mouse</td>
 <td>Novocastra</td>
 <td>NCL-proin-1G4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Proinsulin</td>
 <td>Mouse</td>
 <td>Developmental Studies Hybridoma Bank</td>
 <td>GS-9A8</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Secretagogin</td>
 <td>Rabbit</td>
 <td>Sigma</td>
 <td>HPA 006641</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Smooth muscle actin</td>
 <td>Rabbit</td>
 <td>Abcam</td>
 <td>ab5694</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Somatostatin</td>
 <td>Rabbit</td>
 <td>DAKO</td>
 <td>A0566</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Synaptophysin</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M0776</td>
 <td>Citrate</td>
 </tr>
 <tr>
 <td>Fresh Frozen</td>
 <td>Host species</td>
 <td>Vendor</td>
 <td>Cat. #</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>CD11b</td>
 <td>Rat</td>
 <td>Abcam</td>
 <td>ab6332</td>
 <td>Fix with 4% PF</td>
 </tr>
 <tr>
 <td>CD11c</td>
 <td>Rabbit</td>
 <td>Serotec</td>
 <td>AHP1226</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD25 </td>
 <td>Mouse</td>
 <td>Novus Biological</td>
 <td>NB 600-564</td>
 <td>Use overnight incubation for paraffin sections</td>
 </tr>
 <tr>
 <td>CD94 </td>
 <td>Mouse</td>
 <td>Abcam</td>
 <td>ab61874</td>
 <td>Fix with acetone</td>
 </tr>
 <tr>
 <td>GAD65</td>
 <td>Mouse</td>
 <td>Santa Cruz</td>
 <td>SC-130569</td>
 <td>Fix with 4% PF</td>
 </tr>
 <tr>
 <td>HLA-ABC</td>
 <td>Mouse</td>
 <td>DAKO</td>
 <td>M0736</td>
 <td>Fix with acetone or 4% PF</td>
 </tr>
 </tbody>
</table>
Table 2. Primary antibodies.
<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Aperio Scanscope CS</td>
 <td>Aperio</td>
 <td>&nbsp;</td>
 <td>Digital slide scanner</td>
 </tr>
 <tr>
 <td>DAKO Autostainer Plus</td>
 <td>DAKO</td>
 <td>&nbsp;</td>
 <td>IHC stains</td>
 </tr>
 <tr>
 <td>Label Matrix printing software</td>
 <td>BioCarta</td>
 <td>LM7UP57</td>
 <td>Slide label software</td>
 </tr>
 <tr>
 <td>Leica XL Autostainer</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>H&amp;E stains</td>
 </tr>
 <tr>
 <td>Premium Coverglass</td>
 <td>FisherScientific</td>
 <td>12-548-5J</td>
 <td>Coverslip</td>
 </tr>
 <tr>
 <td>Spectrum Information Manager</td>
 <td>Aperio</td>
 <td>&nbsp;</td>
 <td>Scanner software</td>
 </tr>
 <tr>
 <td>Superfrost Plus slides</td>
 <td>FisherScientific</td>
 <td>12-550-15</td>
 <td>Positively charged slides</td>
 </tr>
 <tr>
 <td>Vegetable steamer</td>
 <td>Black and Decker</td>
 <td>Various</td>
 <td>Antigen retrieval</td>
 </tr>
 <tr>
 <td>Zebra TLP 3742 </td>
 <td>CDWG</td>
 <td>TLP3742</td>
 <td>Slide label printer</td>
 </tr>
 </tbody>
</table>
Table 3. Specific supplies and equipment.

staining protocols for human pancreatic islets

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&amp;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.

Watch this Scientific Journal Video about Staining Protocols for Human Pancreatic Islets at JoVE.com

Staining Protocols for Human Pancreatic Islets

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&amp;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.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

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26.7K Views.  University of Florida. 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.

Video: Staining Protocols for Human Pancreatic Islets

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...