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

Forfatterne takker donorernes familier og de organudtagning involveret i denne forskning og Emily Montgomery, Robert Pietras, Ann Fu, Mitali Agarwal, og Roshan Agarwal for deres eksperthjælp. Dette arbejde blev finansieret af Juvenile Diabetes Research Foundation (MC-T.) til støtte for Netværk for Pankreatisk organdonorer med Diabetes.

1. Microtomy for ufarvede Sektioner <ol><li> Opsæt vandbad og mikrotom som for standard paraffin microtomy. Brug positivt ladede dias og præ-label med sagsnummer, bugspytkirtel region (prøve) og dias nummer. Sted paraffin blok i mikrotomen patronen med kassetten etiketten på venstre side. </li><li> Følg normale microtomy procedurer, afsnit i blok indtil væv ensartet er stødt på. Udarbejdelse af et bånd af serielle sektioner (4 um tykke, 3-6 afhængigt af antallet af oprindelige krævede pletter (se sag materi...

<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>

Standardisering af IHC procedurer er kritisk for billedanalyse, især når der anvendes computer-baserede algoritme i store antal objektglas over tid. Den IHC farvning beskrevet i denne rapport vil gøre det muligt batch analyse af en given donors prøver ved hjælp af en Autostainer inden for en 8-timers arbejdsdag og er ændret fra en tidligere rapport 5. Digitaliserede hele dias billeder af hver farves slide stilles til rådighed for nPOD-tilsluttede efterforskere gennem web-baserede online patologi system...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> 30% Hydrogenperoxid (H2O 2) </td><td> FisherScientific </td><td> H325-500 </td><td> Endogen peroxidase blokerende </td></tr><tr><td> ABC-alkalisk phosphatase (AP) </td><td> Vector </td><td> AK-5000 </td><td> AP konjugat </td></tr><tr><td> Antibody Diluent </td><td> Invitrogen </td><td> 003218 </td><td> Primære antistof </td></tr><tr><td> Avidin blocker </td><td> Vector </td><td> SP-2001 </td><td> Blokere endogent biotin </td></tr><tr><td> Biotinyleret gede-anti-marsvine </td><td> Vector </td><td> BA-7000 </td><td> Sekundært antistof </td></tr><tr><td> Bluing Reagent </td><td> FisherScientific </td><td> 7301 </td><td> Leica autostainer </td></tr><tr><td> Citratbuffer, pH 6,0 </td><td> BioGenex </td><td> HK086-9K </td><td> Antigen hentning </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> Dækglasset mountant </td></tr><tr><td> DAB </td><td> Vector </td><td> SK-4100 </td><td> HRP kromogen </td></tr><tr><td> Deeb </td><td> DAKO </td><td> S2003 </td><td> Endogen AP blokeringsreagens </td></tr><tr><td> Eosin Y Alkoholisk </td><td> FisherScientific </td><td> 71204 </td><td> Leica Autostainer </td></tr><tr><td> Ethanoler-100%, 95%, 90%, 70% </td><td> FisherScientific </td><td> Various </td><td> Afparaffinering og dækglas </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 (alle værtsarter for primære antistoffer) </td><td> Various </td><td> Various </td><td> Negative kontroller til primærvalg </td></tr><tr><td> Liquid Permanent Red (LPR) </td><td> DAKO </td><td> K0640 </td><td> AP kromogen </td></tr><tr><td> Mach2 AP </td><td> Biocare Medicinsk </td><td> MALP521L </td><td> Ged anti-mus AP-konjugat </td></tr><tr><td> Mach2 HRP </td><td> Biocare Medicinsk </td><td> MHRP520L </td><td> Ged anti-mus HRP-konjugat </td></tr><tr><td> Mach2 HRP </td><td> Biocare Medicinsk </td><td> RHRP520L </td><td> Ged anti-mus HRP-konjugat </td></tr><tr><td> Methanol </td><td> FisherScientific </td><td> Various </td><td> H 2 O 2 fortyndingsmiddel </td></tr><tr><td> Normalt gedeserum </td><td> Vector </td><td> S-1000 </td><td> IHC blokeringsreagens </td></tr><tr><td> Sniper </td><td> Biocare Medicinsk </td><td> BS966M </td><td> IHC blokeringsreagens </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> Xylen </td><td> FisherScientific </td><td> Various </td><td> Afparaffinering og dækglas </td></tr></tbody></table> Tabel 1. Specifikke reagenser. <table border="1"><tbody><tr><td> Primært antistof </td><td> Værtsarter </td><td> Vendor </td><td> Kat. # </td><td> Kommentarer og Antigen Hentningsindstillinger </td></tr><tr><td> Paraffin </td><td></td><td></td><td></td><td></td></tr><tr><td> Amylase </td><td> Mus </td><td> Santa Cruz </td><td> SC-46.657 </td><td> Citrat </td></tr><tr><td> Amylin </td><td> Mus </td><td> Serotec </td><td> MCA11267 </td><td> Citrat </td></tr><tr><td> Kulsyreanhydrase 19,9 </td><td> Mus </td><td> Abcam </td><td> ab15146 </td><td></td></tr><tr><td> Caspase-3, spaltet </td><td> Kanin </td><td> Cellesignalering </td><td> 9961 </td><td> Citrat eller Trilogy </td></tr><tr><td> CD20 </td><td> Mus </td><td> DAKO </td><td> M0755 </td><td> Citrat eller Trilogy </td></tr><tr><td> CD3 </td><td> Kanin </td><td> DAKO </td><td> A0452 </td><td> Citrat </td></tr><tr><td> CD34 </td><td> Mus </td><td> Cellesignalering </td><td> 3569 </td><td> Citrat </td></tr><tr><td> CD4 </td><td> Mus </td><td> DAKO </td><td> M7310 </td><td> Citrat </td> &lt;/ Tr&gt; <tr><td> CD45 </td><td> Mus </td><td> DAKO </td><td> M0754 </td><td> Citrat </td></tr><tr><td> CD68 </td><td> Mus </td><td> DAKO </td><td> M0876 </td><td> Citrat </td></tr><tr><td> CD8 </td><td> Mus </td><td> DAKO </td><td> M7103 </td><td> Citrat </td></tr><tr><td> Chromogranin A </td><td> Kanin </td><td> DAKO </td><td> A0430 </td><td> Citrat </td></tr><tr><td> CK17 </td><td> Mus </td><td> DAKO </td><td> M7046 </td><td> Citrat </td></tr><tr><td> CK19 </td><td> Mus </td><td> DAKO </td><td> M0772 </td><td> Citrat </td></tr><tr><td> CK7 </td><td> Mus </td><td> DAKO </td><td> M7018 </td><td> Citrat </td></tr><tr><td> C-peptid </td><td> Kanin </td><td> Cellesignalering </td><td> 4593 </td><td> Citrat </td></tr><tr><td> Foxp3 </td><td> Rotte </td><td> e Bioscience </td><td> 14-4776-80 </td&gt; <td> Citrat </td></tr><tr><td> Ghrelin </td><td> Goat </td><td> Santa Cruz </td><td> SC-10.386 </td><td></td></tr><tr><td> Ghrelin </td><td> Kanin </td><td> Abcam </td><td> ab85104 </td><td></td></tr><tr><td> Glucagon </td><td> Kanin </td><td> DAKO </td><td> A0565 </td><td></td></tr><tr><td> Glucagon </td><td> Mus </td><td> Abcam </td><td> ab10988 </td><td> Citrat </td></tr><tr><td> Glucagon </td><td> Marsvin </td><td> Bachem </td><td> T-5037 </td><td> Citrat </td></tr><tr><td> Glut-1 </td><td> Mus </td><td> Abcam </td><td> ab40084 </td><td> Trilogy </td></tr><tr><td> Glut-2 </td><td> Kanin </td><td> Santa Cruz </td><td> SC-9117 </td><td> Ikke udtrykt i humane øer </td></tr><tr><td> Insulin </td><td> Marsvin </td><td> DAKO </td><td> A0564 </td><td></td></tr> &lt;tr&gt; <td> Ki-67 </td><td> Mus </td><td> DAKO </td><td> M7240 </td><td> Citrat </td></tr><tr><td> MAFA </td><td> Kanin </td><td> Novus Biologisk </td><td> NB400-137A </td><td> Trilogy </td></tr><tr><td> Pancreatisk polypeptid </td><td> Kanin </td><td> Invitrogen </td><td> 18-0043 </td><td> Citrat </td></tr><tr><td> PDX-1 </td><td> Marsvin </td><td> Abcam </td><td> ab47308 </td><td> Citrat eller Trilogy </td></tr><tr><td> Proinsulin </td><td> Mus </td><td> Novocastra </td><td> NCL-proin-1G4 </td><td></td></tr><tr><td> Proinsulin </td><td> Mus </td><td> Developmental Studies Hybridoma Bank </td><td> GS-9A8 </td><td> Citrat </td></tr><tr><td> Secretagogin </td><td> Kanin </td><td> Sigma </td><td> HPA 006641 </td><td> Citrat </td></tr><tr><td> Glat muskel-actin </td><td> Kanin </td><td> Abcam </td><td> ab5694 &lt;/ Td&gt; <td></td></tr><tr><td> Somatostatin </td><td> Kanin </td><td> DAKO </td><td> A0566 </td><td></td></tr><tr><td> Synaptophysin </td><td> Mus </td><td> DAKO </td><td> M0776 </td><td> Citrat </td></tr><tr><td> Frisk Frosset </td><td> Værtsarter </td><td> Vendor </td><td> Kat. # </td><td> Kommentarer </td></tr><tr><td> CD11b </td><td> Rotte </td><td> Abcam </td><td> ab6332 </td><td> Fastgør med 4% PF </td></tr><tr><td> CD11c </td><td> Kanin </td><td> Serotec </td><td> AHP1226 </td><td></td></tr><tr><td> CD25 </td><td> Mus </td><td> Novus Biologisk </td><td> NB 600-564 </td><td> Anvendes inkubation natten for paraffinsnit </td></tr><tr><td> CD94 </td><td> Mus </td><td> Abcam </td><td> ab61874 </td><td> Fix med acetone </td></tr><tr><td> GAD65 </td><td> Mus </td><td> Santa Cruz </td><td> SC-130.569 </td><td> Fastgør med 4% PF </td></tr><tr><td> HLA-ABC </td><td> Mus </td><td> DAKO </td><td> M0736 </td><td> Fix med acetone eller 4% PF </td></tr></tbody></table> Tabel 2 nedenfor. Primære antistoffer. <table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Aperio Scanscope CS </td><td> Aperio </td><td></td><td> Digital dias scanner </td></tr><tr><td> DAKO Autostainer Plus </td><td> DAKO </td><td></td><td> IHC pletter </td></tr><tr><td> Label Matrix udskrivningssoftware </td><td> BioCarta </td><td> LM7UP57 </td><td> Slide Label Software </td></tr><tr><td> Leica XL Autostainer </td><td>Leica </td><td></td><td> H &amp; E pletter </td></tr><tr><td> Premium dækglas </td><td> FisherScientific </td><td> 12-548-5J </td><td> Dækglasset </td></tr><tr><td> Spectrum Information Manager </td><td> Aperio </td><td></td><td> Scanner software </td></tr><tr><td> SuperFrost Plus glider </td><td> FisherScientific </td><td> 12-550-15 </td><td> Positivt ladede dias </td></tr><tr><td> Vegetabilsk damper </td><td> Black og Decker </td><td> Various </td><td> Antigen hentning </td></tr><tr><td> Zebra TLP 3742 </td><td> CDWG </td><td> TLP3742 </td><td> Slide etiketprinter </td></tr></tbody></table> Tabel 3. Specifikke forsyninger og udstyr.

staining protocols for human pancreatic islets

Estimater af ø-området og tal og endokrine celler sammensætning i voksne humane pancreas variere fra adskillige hundrede tusinde til adskillige millioner og beta masse i området fra 500 til 1500 mg 1-3. Med denne kendte heterogenitet, blev en standard behandling og farvning, der er udviklet således, at bugspytkirtlen regioner var klart defineret og holme karakteriseret ved hjælp af strenge histopatologi og immunolokalisering undersøgelser. Standardiserede procedurer for behandling af human pancreas inddrives fra organdonorer er beskrevet i del 1 af denne serie. Bugspytkirtlen forarbejdes til 3 vigtigste regioner (hoved, krop, hale), efterfulgt af tværgående sektioner. Tværsektioner fra bugspytkirtlen hovedet er yderligere opdelt, som angivet baseret på størrelse, og nummereret alfabetisk at betegne underafsnit. Denne standardisering muliggør en fuldstændig tværsnit analyse af hovedet regionen inklusiv uncinate region, som indeholder sammensat øvævscellerprimært af pancreatisk polypeptid celler til haleregionen. Den aktuelle rapport udgør del 2 af denne serie, og beskriver de procedurer, som anvendes til seriel sektionering og histopatologisk karakterisering af pancreas paraffinsnit med en vægt på ø-endokrine celler, replikation, og T-celle-infiltrater. Patologi af pancreas sektioner er beregnet til at karakterisere både exokrine, kanalsystem, og endokrine komponenter. Den exokrine rum er undersøgt for tilstedeværelsen af pancreatitis (aktiv eller kronisk), atrofi, fibrose og fedt samt kanalsystemet, især i relation til tilstedeværelsen af pancreatisk intraductal neoplasi 4. Øer evalueres for morfologi, størrelse og densitet, endokrine celler, inflammation, fibrose, amyloid, og tilstedeværelsen af ​​replikerende eller apoptotiske celler med H &amp; E og IHC pletter. Den sidste komponent er beskrevet i del 2 er levering af farvede slides som digitaliserede hele lysbilleder. De digitaliserede dias er arrangeret af tilfælde og bugspytkirtel region i en online-patologi database at skabe en virtuel biobank. Adgang til denne online samling i øjeblikket i alt til over 200 klinikere og forskere involveret i type 1-diabetes forskning. Den online database indeholder et middel til hurtig og fuldstændig deling af data og for efterforskerne at vælge blokke til paraffin eller frosne serielle sektioner.

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

Staining Protocols for Human Pancreatic Islets

Videoen viser fremgangsmåder til karakterisering af humane pancreasøer med hematoxylin og eosin (H &amp; E) og immunhistokemi (IHC). Pancreatiske sektioner fra hoved, krop og hale regioner farves med både H &amp; E og IHC at bestemme ø endokrine sammensætning (insulin, glucagon og pancreatisk polypeptid), cellereplikation (Ki67) og inflammatoriske infiltrater (H &amp; E, CD3). Den uncinate region er lokaliseret ved hjælp IHC for pancreatisk polypeptid.

Farvning protokoller for Menneskerettigheder bugspytkirteløer

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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Medicine

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Immunohistokemisk farvning af B7-H1 (PD-L1) på paraffinindlejret lysbilleder af adenocarcinom i pancreas Tissue

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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

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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 ...

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Isolation, Kultur og Imaging for Human Føtal pankreascelle Clusters

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

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Lepr<sup&gt; Db</sup&gt; Mus Model af type 2 diabetes: Pancreas Islet Isolering og live-cell 2-Photon Imaging af intakte 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, ...

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Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...