EB, KMB, e HX contribuir igualmente. Este estudo foi financiado pelo NCI SPORE em Cânceres Gastrointestinais P50 CA062924, CA148964 NIH K23, Lustgarten Foundation, Viragh Foundation, ASCO Young Investigator Award, NIH 5T32 CA0090701-28 Formação Grant, Sol Goldman Centro de cancro do pâncreas, e da Fundação Nacional de Pâncreas.

B7-H1/PD-L1, um membro da família B7 de imuno-regulação de superfície celular de proteínas, tem um papel importante na regulação negativa de respostas mediadas por células imunitárias através da sua interacção com o seu receptor, a morte programada-1 (DP -1) 1,2. A sobre-expressão de B7-H1 por células tumorais foi observado em vários cancros humanos, incluindo o melanoma, glioblastoma, e carcinomas do pulmão, mama, cólon, ovário e células renais, e tem sido mostrado para danificar anti-tumorais T- imunidade celular 3-8. Recentemente, a expressão B7-H1 por tecidos de adenocarcinoma do pâncreas foi identificado como um potencial marcador de prognóstico 9,10. Além disso, o bloqueio de B7-H1, em um modelo de rato de cancro pancreático foi mostrado para produzir uma resposta anti-tumor 11. Estes dados sugerem a importância de B7-H1 como um potencial alvo terapêutico. Anticorpos anti-B7-H1 bloqueio são, portanto, está sendo testado em ensaios clínicos para humana múltiplatumores sólidos, incluindo melanoma e cancros do pulmão, do cólon, estômago, rim e pâncreas 12. A fim de, eventualmente, ser capaz de identificar os doentes que irão beneficiar de B7-H1 terapias com alvo, é crítico para investigar a correlação entre a expressão e localização de B7-H1 e a resposta do paciente ao tratamento com os anticorpos B7-H1 bloqueio. Examinando a expressão de B7-H1 em tecidos pancreáticos humanos através de adenocarcinoma imunohistoquímica dará um melhor entendimento de como esta molécula de sinalização co-inibidor contribui para a supressão da imunidade antitumoral em microambiente do tumor. O anticorpo anti-B7-H1 monoclonal (clone 5H1), desenvolvido por Chen e colaboradores demonstrou produzir resultados fiáveis ​​em criocortes de coloração de vários tipos de tecidos neoplásicos humanos 4,8, mas a coloração de lâminas embebidas em parafina tinha sido um desafio até Recentemente, 13-18. Nós desenvolvemos o staini B7-H1ng protocolo para slides incluídos em parafina de tecidos de adenocarcinoma do pâncreas. O protocolo de coloração B7-H1 descrito aqui produz a coloração das membranas e citoplasmática consistente de B7-H1 com pouco fundo. 1. De-parafinization <ol><li> Asse desliza a 55 ° C durante 20 min. </li><li> Em um capuz químico, mergulhe slides em xileno por 10 min. </li><li> Imergir as lâminas em xileno fresco por 10 min. </li><li> Imergir desliza em etanol a 100% durante 5 min. </li><li> Imergir desliza em etanol a 100% fresco durante 5 min. </li><li> Imergir desliza em etanol a 95% durante 5 min. </li><li> Imergir desliza em etanol a 80% durante 5 min. </li><li> Imergir desliza em água desionizada durante 5 min. </li></ol> 2. A recuperação antigênica <ol><li> Imergir as lâminas em tampão Tris-EDTA (pH 9,0) e de calor em uma panela de pressão a 125 ° C durante 30 seg, depois 90 ° C durante 10 seg. </li><li> Deixe esfriar por lâminas de 60 min. </li><li> Imergir as lâminas em TBST por 5 min; REPEmenos duas vezes. </li></ol> 3. Coloração <ol><li> Limpe soluções em volta do tecido nos slides. Marcar um círculo em torno do tecido com uma caneta hidrofóbica. </li><li> Colocar as lâminas numa câmara de humidade e evitar a secagem do tecido na lâmina durante o processo de coloração inteiro. Colocar uma gota (aproximadamente 200 uL ou superior para cobrir a área do tecido completamente) de bloqueio da peroxidase no círculo hidrofóbico sobre a lâmina e incubar no bloqueio da peroxidase, durante 5 min. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. </li><li> Depositar uma gota de um tampão de bloqueio BLOCK ACE em cada lâmina e incubar durante 15 min. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. </li><li> Colocar uma gota de solução de Avidina em cada lâmina e incubar durante 15 min. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. </li><li> Colocar uma gota de solução de biotina em cada lâmina e incubar durante 15 min. </li><li> Lavar slides com TBST, e depois colocar em TBST durante 5 minutos. </li><li> Incubar as lâminas com a diluição de 1:1000 de anticorpo de ratinho anti-B7-H1 monoclonal (clone 5H1) ou de um rato de controlo de isotipo IgG1 κ em 200 ul de TBST, a 4 ° C durante a noite, durante 22 h + / - 1 h. A concentração de anticorpo final em TBST é 2,57 ng / ml. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. Repita duas vezes. </li><li> Incubar as lâminas com um conjugado de biotina anti-ratinho de rato-IgG1 anticorpo secundário, a uma concentração de 1:500 (em 200 ul de solução de cabra 2% de soro) durante 30 minutos. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. Repita duas vezes. </li><li> Depositar uma gota de um complexo de estreptavidina-biotina (deve ser preparada, pelo menos, 30 min antes da sua utilização) em cada lâmina e incubar durante 15 min. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. Repita duas vezes. </li><li> Colocar uma gota de Reagente de Amplificação (diluído 2:1 com TBST) em cada lâmina e incubar durante 4 min. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. Repita duas vezes. </li><li> Colocar uma gota de Estreptavidina-HRP em cada lâmina e incubar durante 15 min. </li><li> Lavar as lâminas com TBST, e depois colocar em TBST durante 5 minutos. Repita duas vezes. </li><li> Colocar uma gota de 3,3-diaminobenzidina (DAB) e substrato cromogénio em cada lâmina e desenvolver a cor durante 2 min. </li><li> Imediatamente após o desenvolvimento de cor, lavagem das lâminas com DDQ 2 O durante 1-2 min. </li><li> Incubar as lâminas com uma gota de hematoxilina durante 90 segundos. </li><li> Lava-se com DDQ 2 O durante 1-2 min. </li><li> Lavar com água corrente e sabão por 1-2 min. </li><li> Lava-se com DDQ 2 O durante 1-2 min. </li></ol> 4. Desidratação e montagem <ol><li> Imergir as lâminas em etanol a 80% durante 3 min. </li><li> Imergir as lâminas em etanol a 95% durante 3 min. </li><li> Imergir as lâminas em etanol a 100% durante 5 min. Repetir. </li><li> Mergulhe as lâminas em xilol por 5 min. Repetir. </li><li> Adicione uma gota deCytoseal 60 para o slide e colocar a lamela. </li></ol> A Tabela 1 enumera os reagentes utilizados nos procedimentos acima descritos.

<ol>
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A coloração imuno-histoquímica de B7-H1 em criocortes tem sido relatada em estudos anteriores para uma variedade de cancros 3,4. No entanto, as amostras de tumor clínicos geralmente só estão disponíveis sob a forma de blocos embebidos em parafina. O anticorpo monoclonal 5H1 mais recentemente tem sido aplicado com sucesso para a coloração de tecidos embebidos em parafina de tumores, incluindo carcinoma de células renais, carcinoma cervical, cancro da bexiga e melanoma 13-18. B7-H1 coloraç...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> CSA Sistema </td><td> Dako </td><td> K1500 </td><td> Inclui os seguintes reagentes mencionados no protocolo: bloqueio da peroxidase, estreptavidina-biotina complexos, reagentes de amplificação, estreptavidina-HRP e DAB substrato eo cromógeno </td></tr><tr><td> Avidina / biotina Blocking Kit </td><td> Vetor </td><td> SP-2001 </td><td></td></tr><tr><td> Bloco ACE </td><td> Serotec </td><td> BUF029 </td><td></td></tr><tr><td> Biotina anti-mouse IgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> IgG1 de ratinho de controlo K Isotype </td><td> eBioscience </td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS (Tris-solução salina tamponada), 10X </td><td> Cellgro </td><td> 46-012 CM- </td><td> 10xTBS contém 80 g / L de NaCl e 24,2 g / L Tirs; Diluído a 1X com ddH2O para lavagens; </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P1379 </td><td> Adicionado 1 ml para 1 L de 1X TBS para fazer TBST </td></tr><tr><td> Alvo de recuperação, 20x </td><td> Celerus Onda </td><td> 014-3000-000 </td><td> Diluída para 1x com DDH 2 O </td></tr><tr><td> 190 Prova Álcool Etílico </td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> 200 Prova Álcool 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> Lamelas </td><td> Corning </td><td> 2940-245 </td><td></td></tr><tr><td> Câmara de umidade </td><td> Sigma </td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Reagentes e equipamentos. </td></tr></tbody></table>

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

B7-H1/PD-L1, um membro da família B7 de imuno-regulação de superfície celular de proteínas, tem um papel importante na regulação negativa de respostas mediadas por células imunitárias através da sua interacção com o seu receptor, a morte programada-1 (DP -1) 1,2. A sobre-expressão de B7-H1 por células tumorais foi observado em vários cancros humanos, incluindo o melanoma, glioblastoma, e carcinomas do pulmão, mama, cólon, ovário e células renais, e tem sido mostrado para danificar anti-tumorais T- imunidade celular 3-8. Recentemente, a expressão B7-H1 por tecidos de adenocarcinoma do pâncreas foi identificado como um potencial marcador de prognóstico 9,10. Além disso, o bloqueio de B7-H1, em um modelo de rato de cancro pancreático foi mostrado para produzir uma resposta anti-tumor 11. Estes dados sugerem a importância de B7-H1 como um potencial alvo terapêutico. Anticorpos anti-B7-H1 bloqueio são, portanto, está sendo testado em ensaios clínicos para multiple tumores sólidos humanos, incluindo melanoma e cancros do pulmão, do cólon, estômago, rim e pâncreas 12. A fim de, eventualmente, ser capaz de identificar os doentes que irão beneficiar de B7-H1 terapias com alvo, é crítico para investigar a correlação entre a expressão e localização de B7-H1 e a resposta do paciente ao tratamento com os anticorpos B7-H1 bloqueio. Examinando a expressão de B7-H1 em tecidos pancreáticos humanos através de adenocarcinoma imunohistoquímica dará um melhor entendimento de como esta molécula de sinalização co-inibidor contribui para a supressão da imunidade antitumoral em microambiente do tumor. O anticorpo anti-B7-H1 monoclonal (clone 5H1), desenvolvido por Chen e colaboradores demonstrou produzir resultados fiáveis ​​em criocortes de coloração de vários tipos de tecidos neoplásicos humanos 4,8, mas a coloração de lâminas embebidas em parafina tinha sido um desafio até Recentemente, 13-18. Temos devfugiu do protocolo de coloração B7-H1 para os slides incluídos em parafina de tecidos de adenocarcinoma do pâncreas. O protocolo de coloração B7-H1 descrito aqui produz a coloração das membranas e citoplasmática consistente de B7-H1 com pouco fundo.

A coloração imuno-histoquímica de sucesso de B7-H1 irá demonstrar a expressão heterogénea de B7-H1 (castanho sinais de desenvolvimento de cor DAB) em adenocarcinoma pancreático. Algumas células de adenocarcinoma do pâncreas têm uma expressão predominantemente membranosas de B7-H1 (Figura 1A) enquanto que outros têm uma expressão ou predominantemente citoplasmática de B7-H1 ou pouca expressão de B7-H1 (Figura 2 e 3). Epitélio pancreático normal duto expressa pouco B7-H1 ...

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) e a sua ligação ao PD-1 fornece um sinal induzida por tumor principal imunossupressor no microambiente do tumor. Uma técnica de coloração imuno-histoquímica para caracterizar a expressão e localização de B7-H1 em adenocarcinoma pancreático é descrito aqui.

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 ...

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 visualizações.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) e a sua ligação ao PD-1 fornece um sinal induzida por tumor principal imunossupressor no microambiente do tumor. Uma técnica de coloração imuno-histoquímica para caracterizar a expressão e localização de B7-H1 em adenocarcinoma pancreático é descrito aqui.

Vídeo: 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 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 ...

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.

Formação de Epithelium próstata humano utilizando Tissue Recombinação de Rodent Urogenital Mesênquima Sinus e células-tronco 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 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 ...

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.

Preparação e Avaliação respirométrico de mitocôndrias isoladas de tecido muscular esquelético Obtido por meio de agulha de biópsia percutânea

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.

Aplicação de anastomose to-end em robótica pancreatectomia 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 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 ...

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.

Rotulagem de imunofluorescência de armadilhas extracelulares de neutrófilos humanos e murinos em tecido embebido em 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.

Enucleação robótica de um insulinoma intrapansático na cabeça pancreatic

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.

Medição do teor de ferro não-heme do tecido usando um ensaio colorimétrico baseado em banhontrolina

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.

Predição de Farmacologia de Rede e Validação Experimental do Mecanismo de Ação do Trichosanthes-Fritillaria thunbergii Contra o Adenocarcinoma Pulmonar