Os autores agradecem as famílias dos doadores e as Organizações de Procura de Órgãos envolvidos na pesquisa e Montgomery Emily, Pietras Robert, Fu Ann, Agarwal Mitali, e Agarwal Roshan para a sua assistência especializada. Este trabalho foi financiado pela Juvenile Diabetes Research Foundation (MC-T.) em apoio da Rede de Doadores de Órgãos do pâncreas com Diabetes.

1. Microtomia para as secções não coradas <ol><li> Configure o banho de água e micrótomo de parafina para microtomia como padrão. Use slides carregados positivamente e pré-label com o número de caso, região do pâncreas (amostra) eo número de slide. Colocar o bloco de parafina no mandril micrótomo com o rótulo da cassete no lado esquerdo. </li><li> Siga os procedimentos normais, microtomia secção em bloco até que o tecido é uniformemente encontrada. Produzir uma fita de cortes seriados (4 mm de espessura, 3-6 dependendo do número de manchas iniciais necessários (ver Formulário Caso Submission, Apêndice 1)). Seções separadas na fita, pegue cada um em ordem, e local em lâminas com ordem numérica com o lápis. Denote se uma seção não é utilizada pela numeração seções na ordem exata. Manter a mesma orientação sobre a lâmina como encontrado no cassete com a etiqueta lâmina orientada para a esquerda. Secar durante a noite à temperatura ambiente, em seguida, prosseguir com a coloração ou de distribuição para os investigadores. </li><li> Ressuperfície eal de bloco de parafina com fina camada de parafina para preservar o tecido e arquivar à temperatura ambiente ou -20 ° C. </li><li> Para microtomia subseqüente, manter a numeração de secções seriadas a cada nível acima. Obter 1 slide adicional e mancha por slide H &amp; E para cada nível mM ~ 100 dentro de cada bloco. </li></ol> 2. H &amp; E Coloração <ol><li> Colocar a lâmina de série primeiro seccionados a partir de cada bloco em um porta-lâminas em seguida, na primeira bandeja na Autostainer. </li><li> Selecione o programa H &amp; E padrão de manchas e proceder como de costume. </li><li> Quando terminar coloração, retire os slides de Autostainer e lugar na capa de lamínulas. </li><li> Lamela utilizando a técnica padrão. Seque bem e rótulo com uma etiqueta impressa (ver secção 12). </li></ol> 3. IHC Set-up <ol><li> Selecione o programa Dako para manchas de casal e os números de entrada de slides. Preparar TBST e citrato de buffers, anticorpos, e Reag outroentos de acordo com volumes indicados (Tabela 1). Carregar o tabuleiro de reagente e as lâminas. A rotação do limpa e linhas de resíduos são programados de acordo com as recomendações do fabricante. </li><li> Se a realização destes ensaios manualmente, preparar volumes estimados. Incubar as lâminas em câmara úmida para evitar lâminas secarem em qualquer etapa. Siga o mesmo procedimento para cada reagentes como quando usado para o Autostainer. </li></ol> 4. IHC desparafinização e recuperação de antígenos <ol><li> Colocar as lâminas em xileno durante 5 minutos. Repetir uma vez. Não permita que as lâminas para secar a qualquer momento subseqüente, até indicado como isso irá resultar em fundo excessivo e coloração pobres. </li><li> Transferir as lâminas etanol a 100% durante 2 minutos. Repetir uma vez. </li><li> Colocar as lâminas em recém-preparada de 3% de H2O 2 em metanol durante 10 minutos. </li><li> Dip lâminas em etanol a 90% e, em seguida, colocar em etanol a 90% durante 3 minutos. </li><li> Transferência desliza paraEtanol a 70% durante 1 minuto. </li><li> Lavar as lâminas em água desionizada. </li><li> Pré-aqueça o vapor e tampão de citrato em vapor durante 5 minutos. </li><li> Adicionar as lâminas tampão citrato no vapor e de vapor durante 30 minutos. </li><li> Transferir imediatamente as lâminas de água e segure até que desliza arrefecer à temperatura ambiente (~ 20 minutos). </li><li> Transferir as lâminas para as prateleiras Dako Autostainer de acordo com mancha seqüência (Figura 1) e executar o programa de dupla marcação. As principais etapas do programa Autostainer são listados para que corridas manuais pode duplicar. </li></ol> 5. Primeiros anticorpos primários (Ki-67, CD3, pâncreas polipéptido) <ol><li> Bloquear as lâminas com solução Sniper durante 15 minutos. Lavar duas vezes com TBST. </li><li> Incubar as lâminas em anticorpo primário durante 30 minutos. Lavar duas vezes com TBST. </li><li> Incubar com Mach2 de cabra anti-rato (Ki-67) ou anti-coelho (CD3, polipéptido pancreático) de rábano (HRP) polímero para 30 minutos. Lavar duas vezes com TBST. </li><li> Aplicar 3,3 diaminobenzidina-tetrahidrocloreto (DAB) a slides durante 4 minutos. Lavar duas vezes com água. </li><li> As lâminas incubadas com polipéptido pancreático são mantidas no Dako quando está a ser ensaiada concorrentemente e TBST utilizado para todos os passos subsequentes ao passo que os restantes lâminas são incubadas com o segundo conjunto de anticorpos primários. Alternativamente, para ensaios manuais, avance para a secção 6. </li></ol> 6. Segundo Anticorpos Primários (insulina, glucagon) <ol><li> Incubar as lâminas em Deeb por 20 minutos. Lavar duas vezes com TBST. </li><li> Bloco com soro de cabra normal a 10% em bloqueador avidina de 20% em TBST (insulina) ou Sniper (glucagon) em TBST durante 20 minutos. Lavar duas vezes com TBST. </li><li> Incubar com insulina ou anticorpo primário glucagon durante 15 minutos. Lavar duas vezes com TBST. </li><li> Insulina: incubar em biotinilado cabra, porco anti-guiné na diluição 1:300 por 30 minutos. Lavar duas vezes com TBST. Incubar com ve padrãoctor reagente ABC-AP por 30 minutos. Lavar duas vezes com TBST. </li><li> O glucagon: incubar em tampão durante durante 30 minutos, seguido por anticorpo de cabra anti-rato de fosfatase alcalina (AP) de polímero durante 30 minutos. Lavar duas vezes com TBST. </li><li> Enquanto as lâminas são incubadas em conjugados, aquecer o tampão de LPR à temperatura ambiente. Adicione 1 gota de líquido vermelho permanente (LPR) por 3 mL de tampão antes do uso imediato e coloque no Dako rack de reagente. Incubar as lâminas em LPC por 4 minutos. Lavar duas vezes com água. </li><li> Incubar as lâminas em hematoxilina por 1 minuto. Lavar duas vezes com água. </li><li> Incubar as lâminas em TBS pH 7,6 por 1 minuto. Lavar duas vezes com água. </li><li> Descarregar slides da DAKO Autostainer. </li><li> Imediatamente desidratar lâminas coradas para polipeptídeo pancreático. Para todas as lâminas coradas duplas, seco durante pelo menos 1 hora, em seguida, desidratar. </li></ol> 7. Desidratação <ol><li> Desliza lugar em etanol a 80% durante 1 minuto. </li><li> Mergulhar as lâminas em etanol a 95%. </li&gt; <li> Segurar as lâminas em etanol a 95% durante 30 segundos. </li><li> Transferir as lâminas em etanol 100% e segure 1 minuto. Repita. </li><li> Dip slides em xileno. </li><li> Mantenha as lâminas em xilol durante 1 minuto. Repita. </li><li> Imediatamente montar lamínulas em lâminas com cytoseal. </li></ol> 8. Rotulagem de slides <ol><li> Digite as informações de identificação do caso incluindo órgão e número do bloco, mancha, data e impressão. Número da seção Serial é opcional para as manchas de slides primeiros de cada bloco. Os níveis subseqüentes são indicados por numeração. </li><li> Coloque etiquetas em slides. </li></ol> 9. Digitalização de slides <ol><li> Coloque lâminas coradas na bandeja do scanner e digitalizar de acordo com a instrumentação. </li><li> Organize os slides por doador e tecido tipo (cabeça do pâncreas, o corpo, cauda, ​​baço). </li><li> Lâminas coradas Arquivo à temperatura ambiente e quaisquer lâminas restantes não coradas a -20 ° C até ser utilizado. </li></ol> 10. RepreseResultados ntative Estes procedimentos de coloração foram desenvolvidas para a Rede JDRF para os doadores de órgãos do pâncreas com Diabetes (nPOD) apresentar a caracterização de base de parafina e frescos blocos congelados. Cada doador tem uma caracterização inicial realizada composta de manchar a 2 quarteirões de cada região do pâncreas (cabeça, corpo e cauda) e do baço (Figura 1, ver também ficha de inscrição caso, Anexo 1). A coloração H &amp; E é conduzida usando um Autostainer programado como para uma instalação clínica com clínico-grade soluções utilizadas por toda parte. Como blocos adicionais dos doadores são seccionadas, cada um tem um slide tomadas para a H &amp; E para mostrar morfologia do tecido. Quando os blocos são seccionados de novo, como para distribuição aos investigadores, os níveis mais profundos também terá lâminas colhidas para representante H e E lâminas para documentar a morfologia todo o bloco de tecido, bem como a numeração secções seriadas em cada nível. Lâminas coradas são rotulados comidentificação caso, a região do pâncreas, número do bloco, mancha e data (Figura 2) e digitalizado para o sistema de gestão de informação em linha patologia. Imagens representativas de um dador de controlo são apresentados na Figura 3 em ambas as ampliações de baixa e alta para mostrar os resultados esperados seguindo este procedimento. A lâmina com manchado polipéptido pancreático (D, H) foi ensaiada em um dia diferente com o ensaio realizado manualmente e mostra reduzida intensidade de coloração do contracorante hematoxilina. As diferenças na intensidade de coloração de anticorpos primários ou contracoloração entre os ensaios é para ser evitado particularmente se usando análise de imagem de outro modo desliza com requerer correcção de cor individual para atingir as áreas mesma célula ou contagens. Cada laboratório deve esperar para otimizar as concentrações de anticorpos como o lote para lote variação e outros reagentes e condições podem afetar a intensidade da coloração e especificidade. Com a aquisição de novas reagents, procedimentos de coloração são validados com conhecidos amostras de controlo positivo e negativo para atingir o mesmo grau de intensidade de manchas como com os reagentes anteriores. Anticorpos adicionais optimizados no núcleo patologia nPOD são fornecidos na Tabela 2, tal como uma fonte de referência e têm sido utilizados para multilabeling imunofluorescência para microscopia confocal. <img src="/files/ftp_upload/4068/4068fig1.jpg" alt="A Figura 1"/> Figura 1. Dako grade de coloração. Este esquemática descreve uma análise de rotina a partir de um dador pâncreas nPOD realizada num Autostainer Dako. Os doadores de nPOD são codificados com um número de identificação de 4 dígitos. Duas bandejas de slides são apresentados com slides organizados pela mancha. Configurações de deslizamento alternados são esperados dependendo do número de lâminas de doadores. Os controlos positivos incluem inclusão de um dador positivo conhecido para os dois manchas duplas e baço doador para o Ki-67 e CD3. Os controlos negativos são duplas e incluem duas slides de um bloco de pâncreas doador incubadas com imunoglobulina (Ig) a partir das espécies hospedeiras dos anticorpos primários e duas lâminas de baço dador para a insulina e glucagon. <img src="/files/ftp_upload/4068/4068fig2.jpg" alt="A Figura 2"/> Figura 2. Lâmina dupla representante manchado. Um slide acabado típico é mostrado com os parâmetros de slides de rotulagem e colocação de tecido antes de escaneamento digital é realizada. <img src="/files/ftp_upload/4068/4068fig3.jpg" alt="A Figura 3"/> Figura 3. Representante H &amp; E e manchas IHC em seções pancreáticas. Seções do pâncreas de um doador de órgãos da fêmea adulta (6096-04 Panhead) foram coradas e scans digitais realizados conforme descrito no protocolo. As imagens foram obtidas usando o programa ImageScope visualização de toda a secção (AD) e de um ilhéu (EH). As intensidades das ilhotas esperados mancha para a insulina, glucagon e polipéptido pancreático sãoobservado para as secções BD como delineia secção D que este bloco cabeça do pâncreas contém uma pequena porção da região uncinado ou lóbulo ventral do pâncreas como todos os ilhéus pancreáticos contêm principalmente polipéptido células positivas. Note-se que a contracoloração de hematoxilina no painel D é muito leve, enquanto a contracoloração de hematoxilina intensidades em B e C são ideais. Painéis EH mostram um ilhéu do lóbulo dorsal com a distribuição esperada de células beta e α-células. A poucas células pancreáticas polipeptídicas são encontrados na porção inferior esquerdo da ilhota (H). A, E-H &amp;E; B, F-Ki67 insulina +; C, G-CD3 + glucagon; D, H pâncreas-polipéptido. AD, 0.2x, EH-12.8x.

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Padronização da IHC procedimentos é crítica para a análise de imagem, especialmente quando se utiliza algoritmos baseados em computador através de um grande número de lâminas ao longo do tempo. O processo de coloração IHQ descritos neste relatório permitirá a análise do lote de amostras de um doador dado, utilizando um Autostainer dentro de um dia de trabalho de 8 horas e são modificadas a partir de um relatório anterior 5. Digitalizadas imagens de slides inteiros de cada slide manchado disponi...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Peróxido de hidrogénio a 30% (H 2 O 2) </td><td> FisherScientific </td><td> H325-500 </td><td> A peroxidase endógena de bloqueio </td></tr><tr><td> ABC-fosfatase alcalina (AP) </td><td> Vetor </td><td> AK-5000 </td><td> AP conjugado </td></tr><tr><td> Diluente anticorpo </td><td> Invitrogen </td><td> 003218 </td><td> O anticorpo primário </td></tr><tr><td> Avidina bloqueador </td><td> Vetor </td><td> SP-2001 </td><td> Bloquear biotina endógena </td></tr><tr><td> Biotinilado de cabra anti-porco da guiné </td><td> Vetor </td><td> BA-7000 </td><td> Anticorpo secundário </td></tr><tr><td> Bluing Reagente </td><td> FisherScientific </td><td> 7301 </td><td> Leica autostainer </td></tr><tr><td> Tampão citrato, pH 6,0 </td><td> Biogenex </td><td> HK086-9K </td><td> Recuperação antigênica </td></tr><tr><td> Clarificador 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> Lamela de montagem </td></tr><tr><td> DAB </td><td> Vetor </td><td> SK-4100 </td><td> HRP cromógeno </td></tr><tr><td> Deeb </td><td> DAKO </td><td> S2003 </td><td> AP endógena bloqueando reagente </td></tr><tr><td> Eosina Y alcoólica </td><td> FisherScientific </td><td> 71204 </td><td> Leica Autostainer </td></tr><tr><td> Etanóis-100%, 95%, 90%, 70% </td><td> FisherScientific </td><td> Vário </td><td> Desparafinização e lamínulas </td></tr><tr><td> Hematoxilina </td><td> DAKO </td><td> S3301 </td><td> Dako Autostainer </td></tr><tr><td> Hematoxilina 7211 </td><td> FisherScientific </td><td> 7211 </td><td> Leica Autostainer </td></tr><tr><td> IgG (todas as espécies de hospedeiros para os anticorpos primários) </td><td> Vário </td><td> Vário </td><td> Os controlos negativos para primárias </td></tr><tr><td> Líquido vermelho permanente (LPR) </td><td> DAKO </td><td> K0640 </td><td> AP cromógeno </td></tr><tr><td> Mach2 AP </td><td> Biocare Medical </td><td> MALP521L </td><td> Cabra anti-rato conjugado AP </td></tr><tr><td> Mach2 HRP </td><td> Biocare Medical </td><td> MHRP520L </td><td> Cabra anti-ratinho HRP </td></tr><tr><td> Mach2 HRP </td><td> Biocare Medical </td><td> RHRP520L </td><td> Cabra anti-ratinho HRP </td></tr><tr><td> Metanol </td><td> FisherScientific </td><td> Vário </td><td> H 2 O 2 diluente </td></tr><tr><td> Soro de cabra normal </td><td> Vetor </td><td> S-1000 </td><td> IHC reagente bloqueador </td></tr><tr><td> Sniper </td><td> Biocare Medical </td><td> BS966M </td><td> IHC reagente bloqueador </td></tr><tr><td> TBST 20X </td><td> ThermoScientific </td><td> TA-999-TT </td><td> IHC tampão </td></tr><tr><td> Trilogia 20x </td><td> Marque celular </td><td> 920p-06 </td><td> Recuperação antigênica </td></tr><tr><td> Xileno </td><td> FisherScientific </td><td> Vário </td><td> Desparafinização e lamínulas </td></tr></tbody></table> Tabela 1. Reagentes específicos. <table border="1"><tbody><tr><td> O anticorpo primário </td><td> Espécies hospedeiras </td><td> Vendedor </td><td> Cat. # </td><td> Comentários e recuperação de antígenos </td></tr><tr><td> Parafina </td><td></td><td></td><td></td><td></td></tr><tr><td> Amilase </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> A anidrase carbónica 19,9 </td><td> Mouse </td><td> Abcam </td><td> ab15146 </td><td></td></tr><tr><td> Caspase-3, clivado </td><td> Coelho </td><td> Sinalização celular </td><td> 9961 </td><td> Citrato ou Trilogy </td></tr><tr><td> CD20 </td><td> Mouse </td><td> DAKO </td><td> M0755 </td><td> Citrato ou Trilogy </td></tr><tr><td> CD3 </td><td> Coelho </td><td> DAKO </td><td> A0452 </td><td> Citrato </td></tr><tr><td> CD34 </td><td> Mouse </td><td> Sinalização celular </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> Coelho </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-péptido </td><td> Coelho </td><td> Sinalização celular </td><td> 4593 </td><td> Citrato </td></tr><tr><td> Foxp3 </td><td> Rato </td><td> e Bioscience </td><td> 14-4776-80 </td&gt; <td> Citrato </td></tr><tr><td> A grelina </td><td> Cabra </td><td> Santa Cruz </td><td> SC-10386 </td><td></td></tr><tr><td> A grelina </td><td> Coelho </td><td> Abcam </td><td> ab85104 </td><td></td></tr><tr><td> Glucagon </td><td> Coelho </td><td> DAKO </td><td> A0565 </td><td></td></tr><tr><td> Glucagon </td><td> Mouse </td><td> Abcam </td><td> ab10988 </td><td> Citrato </td></tr><tr><td> Glucagon </td><td> Cobaia </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> Coelho </td><td> Santa Cruz </td><td> SC-9117 </td><td> Não expressa em ilhotas humanas </td></tr><tr><td> Insulina </td><td> Cobaia </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> Mafa </td><td> Coelho </td><td> Novus Biológica </td><td> NB400-137A </td><td> Trilogia </td></tr><tr><td> Polipeptídeo pancreático </td><td> Coelho </td><td> Invitrogen </td><td> 18-0043 </td><td> Citrato </td></tr><tr><td> PDX-1 </td><td> Cobaia </td><td> Abcam </td><td> ab47308 </td><td> Citrato ou 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> Desenvolvimento de Estudos Hybridoma Banco </td><td> GS-9A8 </td><td> Citrato </td></tr><tr><td> Secretagogin </td><td> Coelho </td><td> Sigma </td><td> HPA 006641 </td><td> Citrato </td></tr><tr><td> Actina de músculo liso </td><td> Coelho </td><td> Abcam </td><td> ab5694 &lt;/ Td&gt; <td></td></tr><tr><td> Somatostatina </td><td> Coelho </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> Fresco Congelado </td><td> Espécies hospedeiras </td><td> Vendedor </td><td> Cat. # </td><td> Comentários </td></tr><tr><td> CD11b </td><td> Rato </td><td> Abcam </td><td> ab6332 </td><td> Fixe com PF 4% </td></tr><tr><td> CD11c </td><td> Coelho </td><td> Serotec </td><td> AHP1226 </td><td></td></tr><tr><td> CD25 </td><td> Mouse </td><td> Novus Biológica </td><td> NB 600-564 </td><td> Use incubação durante a noite para parafina </td></tr><tr><td> CD94 </td><td> Mouse </td><td> Abcam </td><td> ab61874 </td><td> Fixar-se com acetona </td></tr><tr><td> GAD65 </td><td> Mouse </td><td> Santa Cruz </td><td> SC-130.569 </td><td> Fixe com PF 4% </td></tr><tr><td> HLA-ABC </td><td> Mouse </td><td> DAKO </td><td> M0736 </td><td> Fixar-se com acetona ou PF 4% </td></tr></tbody></table> Tabela 2. Anticorpos primários. <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Aperio Scanscope CS </td><td> Aperio </td><td></td><td> Scanner de slides Digital </td></tr><tr><td> Além disso DAKO Autostainer </td><td> DAKO </td><td></td><td> Manchas IHC </td></tr><tr><td> Etiqueta software de impressão Matrix </td><td> BioCarta </td><td> LM7UP57 </td><td> Software de etiqueta de slides </td></tr><tr><td> Leica XL Autostainer </td><td>Leica </td><td></td><td> H &amp; E manchas </td></tr><tr><td> Prêmio lamela </td><td> FisherScientific </td><td> 12-548-5J </td><td> Lamela </td></tr><tr><td> Information Manager Spectrum </td><td> Aperio </td><td></td><td> Software do scanner </td></tr><tr><td> Além disso Superfrost desliza </td><td> FisherScientific </td><td> 12-550-15 </td><td> Lâminas carregadas positivamente </td></tr><tr><td> Vapor vegetal </td><td> Black e Decker </td><td> Vário </td><td> Recuperação antigênica </td></tr><tr><td> Zebra TLP 3742 </td><td> CDWG </td><td> TLP3742 </td><td> Impressora de etiquetas de slides </td></tr></tbody></table> Tabela 3. Suprimentos e equipamentos específicos.

staining protocols for human pancreatic islets

As estimativas de área de ilhotas e os números e composição endócrino célula no pâncreas humano adulto variar de várias centenas de milhar a gamas de massa vários milhões e beta de 500 a 1500 mg 1-3. Com essa heterogeneidade conhecido, um tratamento padrão e procedimento de coloração foi desenvolvido para que as regiões pancreáticas foram claramente definidos e ilhotas caracterizados por histopatologia rigorosa e exames imunolocalização. Procedimentos padronizados para o processamento de pâncreas humano recuperado de doadores de órgãos são descritos na parte 1 desta série. O pâncreas é processado em 3 regiões principais (cabeça, corpo, cauda), seguido de cortes transversais. Secções transversais da cabeça do pâncreas são ainda divididas, tal como indicado com base no tamanho, e numerados em ordem alfabética para denotar subsecções. Esta normalização permite uma análise completa transversal em corte da região da cabeça, incluindo a região que contém uncinado ilhotas compostosprincipalmente de células pancreáticas polipéptido para a região da cauda. O presente relatório compreende parte 2 desta série e descreve os procedimentos utilizados para cortes seriados e caracterização histopatológica das seções pancreáticas de parafina com ênfase em células endócrinas das ilhotas, replicação e de células T infiltrados. Patologia de secções pancreáticas destina-se a caracterizar tanto exócrino, ductular, e componentes endócrinos. O compartimento exócrino é avaliada quanto à presença de pancreatite (activo ou crónica), atrofia, fibrose, e gordura, bem como o sistema de condutas, particularmente em relação à presença de neoplasia pancreática intraductal 4. Ilhotas são avaliadas para a morfologia, tamanho e densidade, células endócrinas, inflamação, fibrose, amilóide, bem como a presença de células replicantes ou apoptótico utilizando H e E e as manchas de IHC. O componente final descrito na parte 2 é o fornecimento da lâmina coradas como digitalizados imagens de diapositivos inteiras. Os slides digitalizados são organizados por caso e região do pâncreas em um banco de dados on-line patologia criar um biobanco virtual. O acesso a esta coleção on-line está actualmente previsto para mais de 200 médicos e cientistas envolvidos na pesquisa do diabetes tipo 1. O banco de dados on-line fornece um meio para a rápida e completa partilha de dados e para os investigadores para selecionar blocos de parafina ou congelados cortes seriados.

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

Staining Protocols for Human Pancreatic Islets

Este vídeo demonstra os procedimentos para caracterização de ilhotas pancreáticas humanas com hematoxilina e eosina (H &amp; E) e imuno-histoquímica (IHQ). Seções pancreáticas de cabeça, corpo e regiões da cauda estão manchadas por tanto H &amp; E e IHC para determinar a composição endócrino ilhéu (insulina, glucagon e polipeptídeo pancreático), a replicação celular (Ki67), e infiltrado inflamatório (H &amp; E, CD3). A região uncinado está localizado usando IHC para polipeptídeo pancreático.

Protocolos de coloração de ilhotas pancreáticas humanas

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 visualizações.  University of Florida. Este vídeo demonstra os procedimentos para caracterização de ilhotas pancreáticas humanas com hematoxilina e eosina (H & E) e imuno-histoquímica (IHQ). Seções pancreáticas de cabeça, corpo e regiões da cauda estão manchadas por tanto H & E e IHC para determinar a composição endócrino ilhéu (insulina, glucagon e polipeptídeo pancreático), a replicação celular (Ki67), e infiltrado inflamatório (H & E, CD3). A região uncinado está localizado...

Vídeo: 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 de Ilhotas Humanos de pacientes parcialmente pancreatectomizado

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

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.

A coloração imuno-histoquímica de B7-H1 (PD-L1) na histológicos em parafina lâminas de tecido adenocarcinoma do pâncreas

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 ...

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.

Procedimentos cirúrgicos para um modelo de rato de Transplante de Fígado parcial ortotópico com reconstrução arterial hepática

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.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

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.

Modelo ortotópico bioluminescente de progressão do câncer de pâncreas

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 imagem de Fetal Humano pancreáticas grupos de células

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 Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

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 transplante de ilhotas pancreáticas no modelo de Mouse

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

Robotic Enucleation of an Intra-Pancreatic Insulinoma in the Pancreatic Head

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses associated with pancreatic resection. Robotic surgery may be used as an alternative for open pancreatic enucleation without compromising dexterity and 3D-vision.
We present the case of a 42-year old woman who presented with sweating, tremor and episodes of hypoglycemia. A fasting test confirmed endogenic insulin overproduction. After inconclusive CT- and MRI imaging, endoscopic ultrasonography showed a hypoechoic lesion, which was fully within the pancreatic head. Although consent was obtained for pancreatoduodenectomy, robotic enucleation seemed feasible. After mobilization, intraoperative ultrasonography was used to identify the lesion and its relation with the pancreatic duct. Dissection was performed using a traction suture, hot shears and bipolar diathermia. A sealant patch was applied for hemostasis and a drain placed. The patient developed a grade B pancreatic fistula for which endoscopic sphincterotomy was performed; the surgical drain could be removed in the outpatient clinic after 20 days. Prospective studies should confirm the short- and long-term benefits of robotic enucleation of insulinomas.

Here, we present a robotic approach to enucleate an insulinoma in the pancreatic head.