EB, KMB, HX y contribuir por igual. Este estudio fue apoyado por el NCI SPORE en cánceres gastrointestinales P50 CA062924, CA148964 NIH K23, Fundación Lustgarten, Fundación Viragh, Premio Joven Investigador ASCO, NIH 5T32 CA0090701-28 Training Grant, Sol Goldman cáncer de páncreas Center y la Fundación Nacional de páncreas.

B7-H1/PD-L1, un miembro de la familia B7 de inmuno-reguladoras proteínas de la superficie celular, juega un papel importante en la regulación negativa de respuestas mediadas por células inmunes a través de su interacción con su receptor, muerte programada-1 (PD -1) 1,2. La sobreexpresión de B7-H1 por células tumorales se ha observado en un número de cánceres humanos, incluyendo melanoma, glioblastoma, y ​​los carcinomas de pulmón, mama, colon, ovario y células renales, y se ha demostrado que es perjudicial anti-tumorales T- inmunidad de células 3-8. Recientemente, B7-H1 expresión por los tejidos de adenocarcinoma pancreático se ha identificado como un marcador pronóstico potencial 9,10. Además, el bloqueo de B7-H1 en un modelo de ratón de cáncer de páncreas se ha demostrado que producen una respuesta anti-tumoral 11. Estos datos sugieren la importancia de B7-H1 como una diana terapéutica potencial. Anti-B7-H1 anticuerpos de bloqueo, por lo tanto están probando en ensayos clínicos para el consumo humano múltipletumores sólidos incluyendo el melanoma y los cánceres de pulmón, colon, riñón, estómago y páncreas 12. Con el fin de ser finalmente capaz de identificar a los pacientes que se beneficiarán de B7-H1 focalización terapias, es crítico para investigar la correlación entre la expresión y localización de B7-H1 y la respuesta del paciente al tratamiento con anticuerpos de bloqueo B7-H1. Examinar la expresión de B7-H1 en tejidos pancreáticos humanos adenocarcinoma mediante inmunohistoquímica dará una mejor comprensión de cómo esta molécula de señalización co-inhibidora contribuye a la supresión de la inmunidad antitumoral en el microentorno del tumor. El anticuerpo anti-B7-H1 monoclonal (clon 5H1) desarrollado por Chen y colaboradores se ha demostrado que produce resultados fiables de tinción en secciones criogénicas de múltiples tipos de tejidos neoplásicos humanos 4,8, pero la tinción de portaobjetos incluidos en parafina había sido un reto hasta recientemente 13-18. Hemos desarrollado el staini B7-H1ng protocolo para las diapositivas incrustadas en parafina de los tejidos adenocarcinoma de páncreas. El protocolo de tinción B7-H1 descrito aquí produce tinción consistente membranosa y citoplásmica de B7-H1 con antecedentes. 1. De-parafinization <ol><li> Bake desliza a 55 ° C durante 20 min. </li><li> En una campana química, sumergir los portaobjetos en xileno durante 10 min. </li><li> Sumerja los portaobjetos en xileno fresco durante 10 minutos. </li><li> Sumergir desliza en 100% de etanol durante 5 min. </li><li> Sumergir desliza en etanol fresco 100% durante 5 min. </li><li> Sumergir desliza en 95% de etanol durante 5 min. </li><li> Sumergir desliza en 80% de etanol durante 5 min. </li><li> Sumergir los portaobjetos con agua desionizada durante 5 min. </li></ol> 2. Antígeno recuperación <ol><li> Sumergir los portaobjetos en tampón Tris-EDTA (pH 9,0) y calor en una olla a presión a 125 ° C durante 30 segundos, luego 90 º C durante 10 sec. </li><li> Deje enfriar diapositivas durante 60 minutos. </li><li> Sumerja los portaobjetos en TBST durante 5 min; Repemenos dos veces. </li></ol> 3. La tinción <ol><li> Limpie soluciones alrededor del tejido en las diapositivas. Marcar un círculo alrededor del tejido con un bolígrafo hidrófobo. </li><li> Colocar los portaobjetos en una cámara de humedad y evitar el secado del tejido en la corredera a lo largo de todo el proceso de tinción. Colocar una gota (aproximadamente 200 l o más para cubrir completamente el área de tejido) de bloquear la peroxidasa dentro del círculo hidrófobo sobre el portaobjetos y portaobjetos durante en el bloque peroxidasa durante 5 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. </li><li> Coloque una gota de tampón de bloqueo BLOCK ACE en cada diapositiva y se incuba durante 15 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. </li><li> Coloque una gota de solución de avidina en cada diapositiva y se incuba durante 15 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. </li><li> Coloque una gota de solución de biotina en cada diapositiva y se incuba durante 15 min. </li><li> Enjuague slides con TBST, y echarlo en TBST durante 5 min. </li><li> Incubar los portaobjetos con dilución 1:1.000 de ratón anti-B7-H1 anticuerpo monoclonal (clon 5H1) o una IgG1 de ratón κ control de isotipo en 200 TBST mu l a 4 ° C durante la noche para 22 hr + / - 1 hr. La concentración final de anticuerpo en TBST es 2,57 g / ml. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. Repita dos veces. </li><li> Incubar los portaobjetos con una rata conjugado con biotina anti-IgG1 de ratón anticuerpo secundario a una concentración de 1:500 (en 200 l solución 2% de suero de cabra) durante 30 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. Repita dos veces. </li><li> Coloque una gota de estreptavidina-biotina (debe estar preparado por lo menos 30 min antes de su uso) en cada portaobjetos y se incuba durante 15 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. Repita dos veces. </li><li> Coloque una gota de reactivo de amplificación (diluido 2:1 con TBST) a cada lado y se incuba durante 4 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. Repita dos veces. </li><li> Coloque una gota de estreptavidina-HRP en cada portaobjetos y se incuba durante 15 min. </li><li> Enjuagar los portaobjetos con TBST, y echarlo en TBST durante 5 min. Repita dos veces. </li><li> Depositar una gota de 3,3-diaminobencidina (DAB) como sustrato y cromógeno en cada diapositiva y desarrollar el color durante 2 min. </li><li> Inmediatamente después del desarrollo del color, lavar los portaobjetos con ddH 2 O durante 1-2 min. </li><li> Incubar los portaobjetos con una gota de hematoxilina durante 90 segundos. </li><li> Lavar con ddH 2 O durante 1-2 min. </li><li> Lavar con agua corriente y jabón durante 1-2 min. </li><li> Lavar con ddH 2 O durante 1-2 min. </li></ol> 4. Deshidratación y montaje <ol><li> Sumergir los portaobjetos en etanol al 80% durante 3 min. </li><li> Sumergir los portaobjetos en etanol al 95% durante 3 min. </li><li> Sumergir los portaobjetos en 100% de etanol durante 5 min. Repetir. </li><li> Sumergir los portaobjetos en xileno durante 5 min. Repetir. </li><li> Añadir una gota deCytoseal 60 a la corredera y el lugar del cubreobjetos. </li></ol> La Tabla 1 enumera los reactivos utilizados en los procedimientos descritos anteriormente.

<ol>
	<li>Flies, D. B., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+B7s:+playing+a+pivotal+role+in+tumor+immunity.">The new B7s: playing a pivotal role in tumor immunity.</a> J. Immunother. 30, 251-260 (2007).</li><li>Dong, H., Zhu, G., Tamada, K., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1,+a+third+member+of+the+B7+family,+co-stimulates+T-cell+proliferation+and+interleukin-10+secretion.">B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion.</a> Nat. Med. 5, 1365-1369 (1999).</li><li>Curiel, T. J., Wei, S., Dong, H., Alvarez, X., Cheng, P., Mottram, P., Krzysiek, R., Knutson, K. 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Med. 8, 793-800 (2002).</li><li>Dong, H., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+pathway+and+its+role+in+the+evasion+of+tumor+immunity.">B7-H1 pathway and its role in the evasion of tumor immunity.</a> J. Mol. Med. (Berl). 81, 281-287 (2003).</li><li>Strome, S. E., Dong, H., Tamura, H., Voss, S. G., Flies, D. B., Tamada, K., Salomao, D., Cheville, J., Hirano, F., Lin, W., Kasperbauer, J. L., Ballman, K. V., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+blockade+augments+adoptive+T-cell+immunotherapy+for+squamous+cell+carcinoma.">B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma.</a> Cancer Res. 63, 6501-6505 (2003).</li><li>Hirano, F., Kaneko, K., Tamura, H., Dong, H., Wang, S., Ichikawa, M., Rietz, C., Flies, D. B., Lau, J. S., Zhu, G., Tamada, K., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+and+PD-1+by+monoclonal+antibodies+potentiates+cancer+therapeutic+immunity.">Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity.</a> Cancer Res. 65, 1089-1096 (2005).</li><li>Thompson, R. H., Gillett, M. D., Cheville, J. C., Lohse, C. M., Dong, H., Webster, W. S., Krejci, K. G., Lobo, J. R., Sengupta, S., Chen, L., Zincke, H., Blute, M. L., Strome, S. E., Leibovich, B. C., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costimulatory+B7-H1+in+renal+cell+carcinoma+patients:+Indicator+of+tumor+aggressiveness+and+potential+therapeutic+target.">Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17174-17179 (2004).</li><li>Geng, L., Huang, D., Liu, J., Qian, Y., Deng, J., Li, D., Hu, Z., Zhang, J., Jiang, G., Zheng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+up-regulated+expression+in+human+pancreatic+carcinoma+tissue+associates+with+tumor+progression.">B7-H1 up-regulated expression in human pancreatic carcinoma tissue associates with tumor progression.</a> J. Cancer Res. Clin. Oncol. 134, 1021-1027 (2008).</li><li>Loos, M., Giese, N. A., Kleeff, J., Giese, T., Gaida, M. M., Bergmann, F., Laschinger, M., WB, M., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+and+regulation+of+the+costimulatory+molecule+B7-H1+in+pancreatic+cancer.">Clinical significance and regulation of the costimulatory molecule B7-H1 in pancreatic cancer.</a> Cancer Lett. 268, 98-109 (2008).</li><li>Okudaira, K., Hokari, R., Tsuzuki, Y., Okada, Y., Komoto, S., Watanabe, C., Kurihara, C., Kawaguchi, A., Nagao, S., Azuma, M., Yagita, H., Miura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+or+B7-DC+induces+an+anti-tumor+effect+in+a+mouse+pancreatic+cancer+model.">Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model.</a> Int. J. Oncol. 35, 741-749 (2009).</li><li>Wang, S., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+cancer+therapies+targeting+CD137+and+B7-H1/PD-1+cosignal+pathways.">Immunobiology of cancer therapies targeting CD137 and B7-H1/PD-1 cosignal pathways.</a> Curr. Top Microbiol. Immunol. 344, 245-267 (2011).</li><li>Thompson, R. H., Webster, W. S., Cheville, J. C., Lohse, C. M., Dong, H., Leibovich, B. C., Kuntz, S. M., Sengupta, S., Kwon, E. D., Blute, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+glycoprotein+blockade:+a+novel+strategy+to+enhance+immunotherapy+in+patients+with+renal+cell+carcinoma.">B7-H1 glycoprotein blockade: a novel strategy to enhance immunotherapy in patients with renal cell carcinoma.</a> Urology. 66, 10-14 (2005).</li><li>Thompson, R. H., Kuntz, S. M., Leibovich, B. C., Dong, H., Lohse, C. M., Webster, W. S., Sengupta, S., Frank, I., Parker, A. S., Zincke, H., Blute, M. L., Sebo, T. J., Cheville, J. C., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+B7-H1+is+associated+with+poor+prognosis+in+renal+cell+carcinoma+patients+with+long-term+follow-up.">Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up.</a> Cancer Res. 66, 3381-3385 (2006).</li><li>Thompson, R. H., Dong, H., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+B7-H1+expression+in+clear+cell+carcinoma+of+the+kidney+for+prognostication+and+therapy.">Implications of B7-H1 expression in clear cell carcinoma of the kidney for prognostication and therapy.</a> Clin. Cancer Res. 13, 709s-715s (2007).</li><li>Karim, R., Jordanova, E. S., Piersma, S. J., Kenter, G. G., Chen, L., Boer, J. M., Melief, C. J., vander Burg, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-expressed+B7-H1+and+B7-DC+in+relation+to+PD-1++T-cell+infiltration+and+survival+of+patients+with+cervical+carcinoma.">Tumor-expressed B7-H1 and B7-DC in relation to PD-1+ T-cell infiltration and survival of patients with cervical carcinoma.</a> Clin. Cancer Res. 15, 6341-6347 (2009).</li><li>Inman, B. A., Sebo, T. J., Frigola, X., Dong, H., Bergstralh, E. J., Frank, I., Fradet, Y., Lacombe, L., Kwon, E. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+study+of+single-agent+anti-programmed+death-1+(MDX-1106)+in+refractory+solid+tumors:+safety,+clinical+activity,+pharmacodynamics,+and+immunologic+correlates.">Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.</a> J. Clin. Oncol. 28, 3167-3175 (2010).</li><li>Wang, L., Ma, Q., Chen, X., Guo, K., Li, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+B7-H1+and+B7-1+expressions+in+pancreatic+carcinoma.">Clinical significance of B7-H1 and B7-1 expressions in pancreatic carcinoma.</a> World J. 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No hay conflictos de interés declarado.

La tinción inmunohistoquímica de B7-H1 en criosecciones se ha informado en estudios anteriores para una variedad de cánceres 3,4. Sin embargo, las muestras de tumores clínicas por lo general sólo están disponibles en forma de bloques incluidos en parafina. El anticuerpo monoclonal 5H1 Más recientemente se ha aplicado con éxito a la tinción de tejidos embebidos en parafina de tumores incluyendo carcinoma de células renales, carcinoma cervical, cáncer de vejiga y melanoma 13-18. B7-H1 en l...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> CSA Sistema </td><td> Dako </td><td> K1500 </td><td> Incluye los siguientes reactivos mencionados en el protocolo: bloque peroxidasa, complejo estreptavidina-biotina, reactivos de amplificación, estreptavidina-HRP y sustrato cromógeno DAB y </td></tr><tr><td> Avidina / biotina Blocking Kit </td><td> Vector </td><td> SP-2001 </td><td></td></tr><tr><td> Bloquear ACE </td><td> Serotec </td><td> BUF029 </td><td></td></tr><tr><td> Biotina anti-ratón IgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> Mouse IgG1 K Control de isotipos </td><td> eBioscience </td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS (Tris-solución salina tamponada), 10X </td><td> Cellgro </td><td> 46-012 CM- </td><td> 10xTBS contiene 80 g / l de NaCl y 24,2 Tirs g / L; Diluido a 1X con ddH2O para lavados; </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P1379 </td><td> 1 ml añadido a 1 L de 1X TBS para hacer TBST </td></tr><tr><td> Target Retrieval, 20x </td><td> Celerus Wave </td><td> 014-3000-000 </td><td> Diluido a 1x con ddH 2 O </td></tr><tr><td> 190 Prueba de Alcohol Etílico </td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> 200 Prueba de alcohol etílico </td><td> PHARMO-Aaper </td><td> 111000200 </td><td></td></tr><tr><td> Xileno </td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td> Hematoxilina </td><td> Richard Allan Scientific- </td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Richard Allan Scientific- </td><td> 8310-4 </td><td></td></tr><tr><td> Cubreobjetos </td><td> Corning </td><td> 2940-245 </td><td></td></tr><tr><td> Humedad cámara </td><td> Sigma </td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 1. Reactivos y equipo. </td></tr></tbody></table>

immunohistochemical staining of b7-h1 (pd-l1) on paraffin-embedded slides of pancreatic adenocarcinoma tissue

B7-H1/PD-L1, un miembro de la familia B7 de inmuno-reguladoras proteínas de la superficie celular, juega un papel importante en la regulación negativa de respuestas mediadas por células inmunes a través de su interacción con su receptor, muerte programada-1 (PD -1) 1,2. La sobreexpresión de B7-H1 por células tumorales se ha observado en un número de cánceres humanos, incluyendo melanoma, glioblastoma, y ​​los carcinomas de pulmón, mama, colon, ovario y células renales, y se ha demostrado que es perjudicial anti-tumorales T- inmunidad de células 3-8. Recientemente, B7-H1 expresión por los tejidos de adenocarcinoma pancreático se ha identificado como un marcador pronóstico potencial 9,10. Además, el bloqueo de B7-H1 en un modelo de ratón de cáncer de páncreas se ha demostrado que producen una respuesta anti-tumoral 11. Estos datos sugieren la importancia de B7-H1 como una diana terapéutica potencial. Anti-B7-H1 anticuerpos de bloqueo, por lo tanto están probando en ensayos clínicos para mulples tumores humanos sólidos, incluyendo el melanoma y los cánceres de pulmón, colon, riñón, estómago y páncreas 12. Con el fin de ser finalmente capaz de identificar a los pacientes que se beneficiarán de B7-H1 focalización terapias, es crítico para investigar la correlación entre la expresión y localización de B7-H1 y la respuesta del paciente al tratamiento con anticuerpos de bloqueo B7-H1. Examinar la expresión de B7-H1 en tejidos pancreáticos humanos adenocarcinoma mediante inmunohistoquímica dará una mejor comprensión de cómo esta molécula de señalización co-inhibidora contribuye a la supresión de la inmunidad antitumoral en el microentorno del tumor. El anticuerpo anti-B7-H1 monoclonal (clon 5H1) desarrollado por Chen y colaboradores se ha demostrado que produce resultados fiables de tinción en secciones criogénicas de múltiples tipos de tejidos neoplásicos humanos 4,8, pero la tinción de portaobjetos incluidos en parafina había sido un reto hasta recientemente 13-18. Tenemos devse fugó el B7-H1 tinción protocolo para las diapositivas incrustadas en parafina de los tejidos adenocarcinoma de páncreas. El protocolo de tinción B7-H1 descrito aquí produce tinción consistente membranosa y citoplásmica de B7-H1 con antecedentes.

Tinción inmunohistoquímica de éxito B7-H1 demostrará la expresión heterogénea de B7-H1 (marrón señales desarrollo del color DAB) en el adenocarcinoma de páncreas. Algunas células de adenocarcinoma pancreático tienen predominantemente membranosa expresión de B7-H1 (Figura 1A) mientras que otras tienen una expresión ya sea predominantemente citoplasmática de B7-H1 o poca expresión de B7-H1 (Figuras 2 y 3). Epitelio del conducto pancreático normal expresa poco B7-H1 <strong...

Watch this Scientific Journal Video about Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue at JoVE.com

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

B7-H1 (PD-L1) y su unión a PD-1 proporcionan una importante inducida por tumor señal inmunosupresora en el microentorno del tumor. Una técnica de tinción inmunohistoquímica para caracterizar la expresión y localización de B7-H1 en el adenocarcinoma pancreático se describe aquí.

La tinción inmunohistoquímica de B7-H1 (PD-L1) en Parafina embebido en diapositivas de tejido pancreático 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 ...

immunohistochemical-staining-b7-h1-pd-l1-on-paraffin-embedded-slides

Research

JoVE Journal

Medicine

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

23.0K vistas.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) y su unión a PD-1 proporcionan una importante inducida por tumor señal inmunosupresora en el microentorno del tumor. Una técnica de tinción inmunohistoquímica para caracterizar la expresión y localización de B7-H1 en el adenocarcinoma pancreático se describe aquí.

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

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolos de tinción de islotes pancreáticos humanos

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

Investigación

Medicina

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

La formación del epitelio prostático humano utilizando tejido recombinación de roedores Urogenital mesénquima seno y células madre humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

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 bioluminiscente de la progresión del cáncer de páncreas

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

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Evaluación de Preparación y respirometría de mitocondrias aisladas de tejido músculo esquelético Obtenidos percutánea Biopsia con aguja

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Aplicación de End-to-end Anastomosis en robótica Pancreatectomía Central

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

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 trasplante de islotes pancreáticos en modelo murino

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

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Etiquetado de inmunofluorescencia de trampas extracelulares de neutrófilos humanos y murinos en tejido incrustado por parafina

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

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.

Enucleación robótica de un insulinoma intrapancrenato en la cabeza pancreática

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Medición del contenido de hierro no hemo en tejidos utilizando un ensayo colorimétrico basado en batofenantrolina

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Predicción de farmacología en red y validación experimental del mecanismo de acción de Trichosanthes-Fritillaria thunbergii contra el adenocarcinoma de pulmón