Gli autori ringraziano le famiglie dei donatori e le Organizzazioni Organ Procurement coinvolti in questa ricerca e Emily Montgomery, Robert Pietras, Ann Fu, Mitali Agarwal, Roshan e Agarwal per la loro assistenza di un esperto. Questo lavoro è stato finanziato dalla Juvenile Diabetes Research Foundation (MC-T.) a sostegno della rete per i donatori di organi del pancreas con diabete.

1. Microtomia per sezioni non macchiate <ol><li> Impostare bagnomaria e microtomo come per microtomia paraffina standard. Utilizzare vetrini con carica positiva e pre-etichetta con il numero di casi, regione pancreas (campione) e il numero di diapositiva. Posto blocco di paraffina nel mandrino microtomo con l&#39;etichetta cassetta sul lato sinistro. </li><li> Seguire le procedure Microtomia normali, sezione nel blocco finché il tessuto è uniformemente incontrato. Produrre un nastro di sezioni seriali (4 micron di spessore, 3-6 a seconda del numero di macchie iniziali richiesti (vedi Modulo per l&#39;invio Case, appendice 1)). Sezioni distinte nella barra multifunzione, pick up in ogni ordine e posto su vetrini con ordine numerata a matita. Indicare se una sezione è inutilizzato da numerazione sezioni in ordine esatto. Mantenere lo stesso orientamento sul vetrino come si trova nella cassetta con l&#39;etichetta slitta orientata verso sinistra. Secco per una notte a temperatura ambiente quindi procedere con colorazione o distribuzione investigatori. </li><li> Reseal superficie del blocco di paraffina con uno strato sottile di paraffina per preservare il tessuto e archivio a temperatura ambiente oa -20 ° C. </li><li> Per microtomia successiva, mantenere la numerazione per le sezioni seriali ad ogni livello come sopra. Ottenere uno scivolo aggiuntivo e macchia da slitta H &amp; E per ogni livello di ~ 100 uM all&#39;interno di ogni blocco. </li></ol> 2. H &amp; E di colorazione <ol><li> Posizionare la prima diapositiva di serie sezionato da ciascun blocco in un rack scorrevole poi nel primo cassetto del Autostainer. </li><li> Selezionare lo standard H &amp; E del programma macchia e procedere come al solito. </li><li> Al termine della colorazione, rimuovere i vetrini da Autostainer e il luogo in cappa per coprioggetto. </li><li> Coprioggetto usando la tecnica standard. Asciugare a fondo e etichetta con un&#39;etichetta stampata (vedere la sezione 12). </li></ol> 3. IHC Set-up <ol><li> Selezionare il programma Dako per le macchie doppi ei numeri di ingresso di diapositive. Preparare buffer TBST e citrato, anticorpi, e Reag altrigenitori in base ai volumi specificati (Tabella 1). Caricare il vassoio dei reagenti e diapositive. Rotazione di pulito e linee di scarico sono programmati secondo le raccomandazioni del fabbricante. </li><li> Se l&#39;esecuzione di questi test manualmente, preparare volumi stimati. Incubare i vetrini in una camera umidificata per evitare vetrini di essiccazione in qualsiasi fase. Seguire la stessa procedura per ogni reagenti esempio per l&#39;Autostainer. </li></ol> 4. IHC Deparaffinizzazione e recupero Antigen <ol><li> Mettere vetrini in xilene per 5 minuti. Ripetere una volta. Non lasciare che i vetrini si asciughino in qualsiasi momento successivo fino indicati come questo si tradurrà in background colorazione eccessiva e poveri. </li><li> Trasferire vetrini al 100% di etanolo per 2 minuti. Ripetere una volta. </li><li> Mettere in vetrini appena preparata 3% H 2 O 2 in metanolo per 10 minuti. </li><li> Immergere i vetrini in etanolo al 90% e quindi inserire nel 90% di etanolo per 3 minuti. </li><li> Trasferimento scorre versoEtanolo 70% per 1 minuto. </li><li> Sciacquare i vetrini in acqua deionizzata. </li><li> Preriscaldare il piroscafo e tampone citrato in vapore per 5 minuti. </li><li> Aggiungere diapositive a citrato buffer nel vapore e il vapore per 30 minuti. </li><li> Trasferire immediatamente scivoli ad acqua e tenere premuto fino a quando scorre raffreddare a temperatura ambiente (~ 20 minuti). </li><li> Trasferire i vetrini con i rack Dako Autostainer secondo macchiare sequenza (Figura 1) ed eseguire il programma di doppia colorazione. Le fasi principali del programma Autostainer sono elencati in modo che piste manuali possono duplicare. </li></ol> 5. Primi Anticorpi primari (Ki-67, CD3, polipeptide pancreatico) <ol><li> Blocca vetrini con la soluzione di Sniper per 15 minuti. Lavare due volte con TBST. </li><li> Incubare i vetrini in anticorpo primario per 30 minuti. Lavare due volte con TBST. </li><li> Incubare con Mach2 capra anti-topo (Ki-67) o anti-coniglio (CD3, polipeptide pancreatico) rafano perossidasi (HRP) polimero per 30 minuti. Lavare due volte con TBST. </li><li> Applicare 3,3-diaminobenzidina tetraidrocloruro (DAB) per i vetrini per 4 minuti. Lavare due volte con acqua. </li><li> Vetrini incubati con il polipeptide pancreatico si svolgono sul Dako quando viene saggiato contemporaneamente e TBST utilizzato per tutti i passi successivi, mentre i vetrini vengono incubati con la seconda serie di anticorpi primari. In alternativa, per le prove manuali, passare alla sezione 6. </li></ol> 6. Secondo Anticorpi Primari (insulina, glucagone) <ol><li> Incubare i vetrini in Deeb per 20 minuti. Lavare due volte con TBST. </li><li> Blocco con 10% siero di capra normale in bloccante avidina 20% in TBST (insulina) o Sniper (glucagone) in TBST per 20 minuti. Lavare due volte con TBST. </li><li> Incubare con insulina o glucagone anticorpo primario per 15 minuti. Lavare due volte con TBST. </li><li> Insulina: vetrini Incubare in biotinilato di capra anti-cavia a 1:300 diluizione per 30 minuti. Lavare due volte con TBST. Incubare con standard di vector ABC-AP reagente per 30 minuti. Lavare due volte con TBST. </li><li> Glucagone: vetrini Incubare in tampone durante per 30 minuti seguiti da capra anti-topo fosfatasi alcalina (AP) polimero per 30 minuti. Lavare due volte con TBST. </li><li> Mentre vetrini vengono incubati in coniugati, riscaldare il buffer LPR a temperatura ambiente. Aggiungere 1 goccia di liquido rosso permanente (LPR) per 3 mL di tampone immediate prima dell&#39;uso e del luogo in rack reagente Dako. Incubare i vetrini in LPC per 4 minuti. Lavare due volte con acqua. </li><li> Incubare i vetrini in ematossilina per 1 minuto. Sciacquare due volte con acqua. </li><li> Incubare i vetrini in TBS pH 7,6 per 1 minuto. Sciacquare due volte con acqua. </li><li> Scaricare diapositive da DAKO Autostainer. </li><li> Immediatamente disidratare vetrini colorati per il polipeptide pancreatico. Per tutte le diapositive doppie colorate, asciutto per almeno 1 ora, quindi disidratano. </li></ol> 7. Disidratazione <ol><li> Posto scorre in 80% etanolo per 1 minuto. </li><li> Immergere i vetrini in etanolo al 95%. </li&gt; <li> Tenere i vetrini in etanolo al 95% per 30 secondi. </li><li> Trasferire i vetrini al 100% di etanolo e tenere premuto 1 minuto. Ripeti. </li><li> Immergere i vetrini in xilene. </li><li> Tenere vetrini in xilene per 1 minuto. Ripeti. </li><li> Immediatamente montare su vetrini coprioggetti usando cytoseal. </li></ol> 8. Diapositiva Labeling <ol><li> Immettere le informazioni di identificazione caso compreso il numero di organi e di blocco, macchia, e la data e la stampa. Numero della sezione di serie è facoltativa per le macchie prima diapositiva di ciascun blocco. I livelli successivi sono indicati con numerazione. </li><li> Posizionare le etichette sulle diapositive. </li></ol> 9. Scansione diapositive <ol><li> Mettere vetrini colorati nel vassoio scanner e la scansione in base alla strumentazione. </li><li> Organizzare le diapositive per tipo di donatore e dei tessuti (testa del pancreas, corpo, coda, milza). </li><li> Vetrini colorati archivio a temperatura ambiente e le diapositive rimanenti non colorati a -20 ° C fino all&#39;uso. </li></ol> 10. RAPPRESERisultati ntative Queste procedure di colorazione sono stati sviluppati per la Rete JDRF per i donatori di organi del pancreas con diabete (nPOD) per fornire la caratterizzazione di base di paraffina e freschi blocchi congelati. Ogni donatore ha una caratterizzazione di base eseguita composto di colorazione a 2 isolati da ogni regione del pancreas (testa, corpo e coda) e la milza (Figura 1, vedi anche modulo di invio caso, appendice 1). La colorazione H &amp; E è condotta utilizzando un Autostainer programmato come per una struttura clinico-clinica con soluzioni di qualità utilizzati in tutto. Come blocchi donatori supplementari sezionate, ognuna ha una diapositiva preso per H &amp; E per mostrare la morfologia dei tessuti. Quando i blocchi sono sezionate volta, come per la distribuzione agli investigatori, i livelli più profondi avrà anche diapositive riprese il rappresentante H &amp; slides E per documentare la morfologia del tessuto l&#39;intero blocco così come numerazione sezioni seriali ad ogni livello. Vetrini colorati sono etichettati condell&#39;individuazione dei casi, regione pancreas, numero di blocco, macchia e la data (Figura 2) e nel sistema di scansione on-line patologia gestione delle informazioni. Immagini rappresentative di un donatore di controllo sono mostrati nella Figura 3 sia a basso ingrandimento e alta per mostrare i risultati attesi seguendo questa procedura. Il tinto diapositiva con il polipeptide pancreatico (D, H) è stato analizzato in un giorno diverso con il test eseguito manualmente e mostra ridotta intensità di colorazione del contrasto ematossilina. Le differenze di intensità di colorazione anticorpi primari o di contrasto tra saggi deve essere evitato soprattutto se utilizzando l&#39;analisi dell&#39;immagine scorre altrimenti con richiedono correzione del colore individuale per ottenere le aree stessa cella o conteggi. Ogni laboratorio deve aspettarsi di ottimizzare le concentrazioni anticorpali da lotto a lotto variazione e altri reagenti e le condizioni possono influenzare l&#39;intensità di colorazione e specificità. Con l&#39;acquisizione di nuove REAsignori, le procedure di colorazione vengono convalidati con noti campioni di controllo positivi e negativi per raggiungere lo stesso grado di intensità macchia con reagenti precedenti. Gli anticorpi ottimizzati nel nucleo nPOD patologia sono riportati nella Tabella 2 come fonte di riferimento e sono stati utilizzati per multilabeling saggi di immunofluorescenza per microscopia confocale. <img src="/files/ftp_upload/4068/4068fig1.jpg" alt="Figura 1"/> Figura 1. Griglia di colorazione Dako. Questo schema raffigura una analisi di routine da un donatore di pancreas nPOD eseguita su un Autostainer Dako. I donatori nPOD sono codificati con un numero a 4 cifre di identificazione. Due vassoi di diapositive vengono visualizzati con diapositive organizzate dalla macchia. Configurazioni di scorrimento alternativi sono previsti a seconda del numero di diapositive donatori. I controlli positivi comprendono l&#39;inclusione di un donatore nota positiva per le due macchie doppie e milza dei donatori per la Ki-67 e CD3. I controlli negativi sono di due tipi e comprendono due slides da un blocco donatore pancreas incubati con immunoglobuline (Ig) dalle specie ospite degli anticorpi primari e due scivoli dalla milza dei donatori per l&#39;insulina e il glucagone. <img src="/files/ftp_upload/4068/4068fig2.jpg" alt="Figura 2"/> Figura 2. Rappresentante doppio slide colorate. Una diapositiva finito tipica è mostrata con i parametri di etichettatura di diapositive e di collocamento dei tessuti prima della scansione digitale viene eseguita. <img src="/files/ftp_upload/4068/4068fig3.jpg" alt="Figura 3"/> Figura 3. Rappresentante H &amp; E e le macchie IHC nelle sezioni pancreatiche. Sezioni da parte del pancreas di un donatore adulto organo femminile (6096-04 Panhead) erano macchiati e scansioni digitali eseguite come descritto nel protocollo. Le immagini sono state ottenute utilizzando il programma ImageScope visualizzazione della intera sezione (AD) e da un isolotto (EH). Le intensità delle isole attesi macchia di insulina, glucagone e polipeptide pancreatico sonoosservati per sezioni BD come sezione D che delimita il blocco testa pancreas contiene una piccola porzione della regione uncinato o lobo ventrale pancreatico come tutte le isole pancreatiche contengono principalmente polipeptide cellule positive. Si noti che il contrasto in ematossilina pannello D è troppo leggero, mentre l&#39;intensità di contrasto in ematossilina B e C sono ottimali. Pannelli EH mostrano un isolotto dal lobo dorsale con la distribuzione prevista dei beta-cellule e α-cellule. A cellule pancreatiche pochi polipeptidiche si trovano nella parte inferiore sinistra della isolotto (H). A, E-H &amp;E; B, F-Ki67 + Insulina, C, G-CD3 + glucagone, D, H-polipeptide pancreatico. AD, 0.2x, EH-12.8x.

<ol>
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Standardizzazione delle procedure IHC è fondamentale per l&#39;analisi delle immagini, in particolare quando si utilizzano computer basati su algoritmi in un gran numero di diapositive nel corso del tempo. La procedura di colorazione IHC descritto nella presente relazione permetterà un&#39;analisi lotto di campioni di un donatore data, utilizzando uno Autostainer all&#39;interno di una giornata di 8 ore di lavoro e vengono modificati da una relazione precedente 5. Immagini delle diapositive digitalizzate in...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 30% perossido di idrogeno (H 2 O 2) </td><td> FisherScientific </td><td> H325-500 </td><td> Perossidasi endogena blocco </td></tr><tr><td> ABC-fosfatasi alcalina (AP) </td><td> Vettore </td><td> AK-5000 </td><td> AP coniugato </td></tr><tr><td> Anticorpo Diluente </td><td> Invitrogen </td><td> 003218 </td><td> Anticorpo primario </td></tr><tr><td> Avidina blocker </td><td> Vettore </td><td> SP-2001 </td><td> Blocca biotina endogena </td></tr><tr><td> Biotinilato di capra anti-cavia </td><td> Vettore </td><td> BA-7000 </td><td> Anticorpo secondario </td></tr><tr><td> Reagente blu </td><td> FisherScientific </td><td> 7301 </td><td> Leica autostainer </td></tr><tr><td> Tampone citrato, pH 6,0 </td><td> BioGenex </td><td> HK086-9K </td><td> Antigen recupero </td></tr><tr><td> Chiarificatore 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 mezzo di montaggio </td></tr><tr><td> DAB </td><td> Vettore </td><td> SK-4100 </td><td> HRP cromogeno </td></tr><tr><td> Deeb </td><td> DAKO </td><td> S2003 </td><td> Endogena AP blocco reattivo </td></tr><tr><td> Eosina alcolica Y </td><td> FisherScientific </td><td> 71204 </td><td> Leica Autostainer </td></tr><tr><td> Etanoli-100%, 95%, 90%, 70% </td><td> FisherScientific </td><td> Vario </td><td> Deparaffinizzazione e coprioggetto </td></tr><tr><td> Ematossilina </td><td> DAKO </td><td> S3301 </td><td> Dako Autostainer </td></tr><tr><td> Ematossilina 7211 </td><td> FisherScientific </td><td> 7211 </td><td> Leica Autostainer </td></tr><tr><td> IgG (tutte le specie di accoglienza per gli anticorpi primari) </td><td> Vario </td><td> Vario </td><td> I controlli negativi per le primarie </td></tr><tr><td> Liquid Permanent Red (LPR) </td><td> DAKO </td><td> K0640 </td><td> AP cromogeno </td></tr><tr><td> Mach2 AP </td><td> Biocare Medical </td><td> MALP521L </td><td> Capra anti-topo coniugato AP </td></tr><tr><td> Mach2 HRP </td><td> Biocare Medical </td><td> MHRP520L </td><td> Capra anti-topo coniugato HRP </td></tr><tr><td> Mach2 HRP </td><td> Biocare Medical </td><td> RHRP520L </td><td> Capra anti-topo coniugato HRP </td></tr><tr><td> Metanolo </td><td> FisherScientific </td><td> Vario </td><td> H 2 O 2 diluente </td></tr><tr><td> Siero di capra normale </td><td> Vettore </td><td> S-1000 </td><td> IHC blocco reattivo </td></tr><tr><td> Cecchino </td><td> Biocare Medical </td><td> BS966M </td><td> IHC blocco reattivo </td></tr><tr><td> TBST 20X </td><td> ThermoScientific </td><td> TA-999-TT </td><td> IHC tampone </td></tr><tr><td> Triology 20x </td><td> Cella Marque </td><td> 920P-06 </td><td> Antigen Retrieval </td></tr><tr><td> Xilene </td><td> FisherScientific </td><td> Vario </td><td> Deparaffinizzazione e coprioggetto </td></tr></tbody></table> Tabella 1. Reagenti specifici. <table border="1"><tbody><tr><td> Anticorpo primario </td><td> Host specie </td><td> Venditore </td><td> Cat. # </td><td> Commenti e recupero Antigen </td></tr><tr><td> Paraffina </td><td></td><td></td><td></td><td></td></tr><tr><td> Amilasi </td><td> Mouse </td><td> Santa Cruz </td><td> SC-46657 </td><td> Citrato </td></tr><tr><td> Amylin </td><td> Mouse </td><td> Serotec </td><td> MCA11267 </td><td> Citrato </td></tr><tr><td> L&#39;anidrasi carbonica 19,9 </td><td> Mouse </td><td> Abcam </td><td> ab15146 </td><td></td></tr><tr><td> Caspasi-3, scisso </td><td> Coniglio </td><td> Segnalazione cellulare </td><td> 9961 </td><td> Citrato o Trilogy </td></tr><tr><td> CD20 </td><td> Mouse </td><td> DAKO </td><td> M0755 </td><td> Citrato o Trilogy </td></tr><tr><td> CD3 </td><td> Coniglio </td><td> DAKO </td><td> A0452 </td><td> Citrato </td></tr><tr><td> CD34 </td><td> Mouse </td><td> Segnalazione cellulare </td><td> 3569 </td><td> Citrato </td></tr><tr><td> CD4 </td><td> Mouse </td><td> DAKO </td><td> M7310 </td><td> Citrato </td> &lt;/ Tr&gt; <tr><td> CD45 </td><td> Mouse </td><td> DAKO </td><td> M0754 </td><td> Citrato </td></tr><tr><td> CD68 </td><td> Mouse </td><td> DAKO </td><td> M0876 </td><td> Citrato </td></tr><tr><td> CD8 </td><td> Mouse </td><td> DAKO </td><td> M7103 </td><td> Citrato </td></tr><tr><td> Cromogranina A </td><td> Coniglio </td><td> DAKO </td><td> A0430 </td><td> Citrato </td></tr><tr><td> CK17 </td><td> Mouse </td><td> DAKO </td><td> M7046 </td><td> Citrato </td></tr><tr><td> CK19 </td><td> Mouse </td><td> DAKO </td><td> M0772 </td><td> Citrato </td></tr><tr><td> CK7 </td><td> Mouse </td><td> DAKO </td><td> M7018 </td><td> Citrato </td></tr><tr><td> C-peptide </td><td> Coniglio </td><td> Segnalazione cellulare </td><td> 4593 </td><td> Citrato </td></tr><tr><td> Foxp3 </td><td> Ratto </td><td> e Bioscience </td><td> 14-4776-80 </td&gt; <td> Citrato </td></tr><tr><td> Grelina </td><td> Capra </td><td> Santa Cruz </td><td> SC-10386 </td><td></td></tr><tr><td> Grelina </td><td> Coniglio </td><td> Abcam </td><td> ab85104 </td><td></td></tr><tr><td> Glucagone </td><td> Coniglio </td><td> DAKO </td><td> A0565 </td><td></td></tr><tr><td> Glucagone </td><td> Mouse </td><td> Abcam </td><td> ab10988 </td><td> Citrato </td></tr><tr><td> Glucagone </td><td> Cavia </td><td> Bachem </td><td> T-5037 </td><td> Citrato </td></tr><tr><td> GLUT-1 </td><td> Mouse </td><td> Abcam </td><td> ab40084 </td><td> Trilogia </td></tr><tr><td> GLUT-2 </td><td> Coniglio </td><td> Santa Cruz </td><td> SC-9117 </td><td> Non espresso in isole umane </td></tr><tr><td> Insulina </td><td> Cavia </td><td> DAKO </td><td> A0564 </td><td></td></tr> &lt;tr&gt; <td> Ki-67 </td><td> Mouse </td><td> DAKO </td><td> M7240 </td><td> Citrato </td></tr><tr><td> CFP </td><td> Coniglio </td><td> Novus biologica </td><td> NB400-137A </td><td> Trilogia </td></tr><tr><td> Polipeptide pancreatico </td><td> Coniglio </td><td> Invitrogen </td><td> 18-0043 </td><td> Citrato </td></tr><tr><td> PDX-1 </td><td> Cavia </td><td> Abcam </td><td> ab47308 </td><td> Citrato o Trilogy </td></tr><tr><td> Proinsulina </td><td> Mouse </td><td> Novocastra </td><td> NCL-proin-1G4 </td><td></td></tr><tr><td> Proinsulina </td><td> Mouse </td><td> Developmental Studies ibridoma Bank </td><td> GS-9A8 </td><td> Citrato </td></tr><tr><td> Secretagogin </td><td> Coniglio </td><td> Sigma </td><td> HPA 006641 </td><td> Citrato </td></tr><tr><td> Actina del muscolo liscio </td><td> Coniglio </td><td> Abcam </td><td> ab5694 &lt;/ Td&gt; <td></td></tr><tr><td> Somatostatina </td><td> Coniglio </td><td> DAKO </td><td> A0566 </td><td></td></tr><tr><td> Sinaptofisina </td><td> Mouse </td><td> DAKO </td><td> M0776 </td><td> Citrato </td></tr><tr><td> Freschi congelati </td><td> Host specie </td><td> Venditore </td><td> Cat. # </td><td> Comments </td></tr><tr><td> CD11b </td><td> Ratto </td><td> Abcam </td><td> ab6332 </td><td> Fissare con il 4% PF </td></tr><tr><td> CD11c </td><td> Coniglio </td><td> Serotec </td><td> AHP1226 </td><td></td></tr><tr><td> CD25 </td><td> Mouse </td><td> Novus biologica </td><td> NB 600-564 </td><td> Utilizzare una notte di incubazione per le sezioni di paraffina </td></tr><tr><td> CD94 </td><td> Mouse </td><td> Abcam </td><td> ab61874 </td><td> Fissare con acetone </td></tr><tr><td> GAD65 </td><td> Mouse </td><td> Santa Cruz </td><td> SC-130569 </td><td> Fissare con il 4% PF </td></tr><tr><td> HLA-ABC </td><td> Mouse </td><td> DAKO </td><td> M0736 </td><td> Fissare con acetone o 4% PF </td></tr></tbody></table> Tabella 2. Anticorpi primari. <table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Aperio Scanscope CS </td><td> Aperio </td><td></td><td> Diapositive scanner digitale </td></tr><tr><td> DAKO Autostainer Plus </td><td> DAKO </td><td></td><td> IHC macchie </td></tr><tr><td> Label Matrix software di stampa </td><td> BioCarta </td><td> LM7UP57 </td><td> Presentazione dell&#39;etichetta del software </td></tr><tr><td> Leica XL Autostainer </td><td>Leica </td><td></td><td> H &amp; E le macchie </td></tr><tr><td> Premium vetrino </td><td> FisherScientific </td><td> 12-548-5J </td><td> Coverslip </td></tr><tr><td> Spectrum Information Manager </td><td> Aperio </td><td></td><td> Scanner software </td></tr><tr><td> SuperFrost Plus scivola </td><td> FisherScientific </td><td> 12-550-15 </td><td> Vetrini con carica positiva </td></tr><tr><td> Verdure a vapore </td><td> Black and Decker </td><td> Vario </td><td> Antigen recupero </td></tr><tr><td> Zebra TLP 3742 </td><td> CDWG </td><td> TLP3742 </td><td> Diapositiva stampante di etichette </td></tr></tbody></table> Tabella 3. 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staining protocols for human pancreatic islets

Le stime di superficie delle isole e dei numeri e la composizione delle cellule endocrine nel pancreas umano adulto varia da alcune centinaia di migliaia di intervalli di massa diversi milioni di beta e da 500 a 1-3 mg 1500. Con questa eterogeneità noto, un trattamento standard e procedura di colorazione è stato sviluppato in modo che le regioni del pancreas sono stati chiaramente definiti e isolotti caratterizzate con rigorosa istopatologia ed esami immunolocalizzazione. Procedure standardizzate per l&#39;elaborazione di pancreas umano recuperati da donatori di organi sono descritte nella parte 1 di questa serie. Il pancreas è trasformato in 3 regioni principali (testa, corpo, coda) seguiti da sezioni trasversali. Sezioni trasversali della testa del pancreas sono ulteriormente suddivise, come indicato in base alle dimensioni, e numerati in ordine alfabetico per indicare sottosezioni. Questa standardizzazione permette una completa analisi sezione trasversale della regione della testa, compresa la regione uncinato che contiene composti isolottiprincipalmente da cellule pancreatiche polipeptide regione della coda. La presente relazione comprende parte 2 di questa serie e descrive le procedure utilizzate per il sezionamento seriale e caratterizzazione istopatologica delle sezioni di paraffina del pancreas con un&#39;enfasi sulle cellule endocrine insulari, la replica e T-cellule si infiltra. Patologia delle sezioni pancreatiche è destinata a caratterizzare sia esocrina, dei dotti, e componenti endocrine. Il vano esocrina viene valutato per la presenza di pancreatite (attivo o cronica), atrofia, fibrosi, e grasso, così come il sistema di condotto, in particolare in relazione alla presenza di neoplasia pancreatica intraduttale 4. Le isole vengono valutati per la morfologia, le dimensioni e densità, cellule endocrine, infiammazione, fibrosi, amiloide, e la presenza di cellule replica o apoptotici con H &amp; E e le macchie IHC. Il componente finale descritta nella parte 2 è la fornitura della diapositiva macchiatas come immagini delle diapositive digitalizzate intere. Le diapositive digitalizzate sono organizzati per caso e regione pancreas in un database online patologia la creazione di una biobanca virtuale. L&#39;accesso a questa raccolta online è attualmente previsto a oltre 200 medici e scienziati coinvolti nella ricerca sul diabete di tipo 1. Il database online fornisce un mezzo per la rapida e completa condivisione dei dati e per i ricercatori per selezionare i blocchi di paraffina o congelati sezioni seriali.

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

Staining Protocols for Human Pancreatic Islets

Questo video mostra le procedure per la caratterizzazione di isole pancreatiche umane utilizzando ematossilina ed eosina (H &amp; E) e di immunoistochimica (IHC). Sezioni del pancreas di testa, corpo, e le regioni di coda sono macchiati sia da H &amp; E e IHC per determinare la composizione delle isole endocrine (insulina, glucagone e polipeptide pancreatico), la replicazione cellulare (Ki67), e infiltrati infiammatori (H &amp; E, CD3). La regione uncinato è localizzato usando IHC per il polipeptide pancreatico.

Protocolli di colorazione di isole pancreatiche umane

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

staining-protocols-for-human-pancreatic-islets

Research

JoVE Journal

Medicine

26.7K Views.  University of Florida. Questo video mostra le procedure per la caratterizzazione di isole pancreatiche umane utilizzando ematossilina ed eosina (H & E) e di immunoistochimica (IHC). Sezioni del pancreas di testa, corpo, e le regioni di coda sono macchiati sia da H & E e IHC per determinare la composizione delle isole endocrine (insulina, glucagone e polipeptide pancreatico), la replicazione cellulare (Ki67), e infiltrati infiammatori (H & E, CD3). La regione uncinato è localizzato ...

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.

Isolamento di isole umane da pazienti parzialmente Pancreatectomized

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

Ricerca

Medicina

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.

Colorazione immunoistochimica di B7-H1 (PD-L1) in paraffina incorporate diapositive di tessuto pancreatico Adenocarcinoma

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.

Isolotti roditori Valutazione di replica e la funzione delle cellule beta in Adenovirally-trasdotte Isolati

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.

Procedure chirurgiche per un modello di Rat parziale trapianto di fegato con la ricostruzione arteriosa epatica

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.

Protocollo migliorato per microdissezione laser di isole pancreatiche umane da campioni chirurgici

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.

Bioluminescente modello ortotopico di cancro al pancreas Progressione

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.

Isolamento, Cultura e Imaging di Human Fetal pancreatici gruppi di cellule

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.

LEPR<sup&gt; Db</sup&gt; Mouse Modello di diabete di tipo 2: pancreas isolamento Islet e live-cella 2-Photon Imaging di isolotti Intact

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.

Intraportal trapianto di isole pancreatiche nel modello del topo

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