Name: staining protocols for human pancreatic islets
Uploaded: 2012-05-23T13:00:00
Description: Watch this Scientific Journal Video about Staining Protocols for Human Pancreatic Islets at JoVE.com

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 C...

<ol>
	<li>In't Veld, P., Marichal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+anatomy+of+the+human+islet+of+Langerhans.">Microscopic anatomy of the human islet of Langerhans.</a> Adv. Exp. Med. Biol. 654, 1-19 (2010).</li><li>Matveyenko, A. V., Butler, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+beta-cell+mass+and+diabetes+onset.">Relationship between beta-cell mass and diabetes onset.</a> Diabetes Obes. Metab. 10, 23-31 (2008).</li><li>Saisho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+volumes+in+humans+from+birth+to+age+one+hundred+taking+into+account+sex,+obesity,+and+presence+of+type-2+diabetes.">Pancreas volumes in humans from birth to age one hundred taking into account sex, obesity, and presence of type-2 diabetes.</a> Clin. Anat. 20, 933-942 (2007).</li><li>Hruban, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+intraepithelial+neoplasia:+a+new+nomenclature+and+classification+system+for+pancreatic+duct+lesions.">Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions.</a> Am. J. Surg. Pathol. 25, 579-586 (2001).</li><li>Campbell-Thompson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+adenocarcinoma+patients+with+localised+chronic+severe+pancreatitis+show+an+increased+number+of+single+beta+cells,+without+alterations+in+fractional+insulin+area.">Pancreatic adenocarcinoma patients with localised chronic severe pancreatitis show an increased number of single beta cells, without alterations in fractional insulin area.</a> Diabetologia. 52, 262-270 (2009).</li><li>Meier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+replication+is+the+primary+mechanism+subserving+the+postnatal+expansion+of+beta-cell+mass+in+humans.">Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans.</a> Diabetes. 57, 1584-1594 (2008).</li><li>Rowe, P. A., Campbell-Thompson, M. L., Schatz, D. A., Atkinson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreas+in+human+type+1+diabetes.">The pancreas in human type 1 diabetes.</a> Semin. Immunopathol. 33, 29-43 (2011).</li><li>Veld, I. n. '. t., P, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulitis+in+human+type+1+diabetes:+The+quest+for+an+elusive+lesion.">Insulitis in human type 1 diabetes: The quest for an elusive lesion.</a> Islets. 3, 131-138 (2011).</li><li>van Belle, T. L., Coppieters, K. T., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes:+etiology,+immunology,+and+therapeutic+strategies.">Type 1 diabetes: etiology, immunology, and therapeutic strategies.</a> Physiol Rev. 91, 79-118 (2011).</li></ol>

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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