EBは、KMB、とHXは均等に貢献しています。本研究では、消化器癌P50 CA062924、NIHのK23 CA148964、ラストガーテン財団、Viragh財団、ASCOの若手研究者賞は、NIH 5T32 CA0090701-28トレーニンググラント、ソルゴールドマン膵臓癌センター、国立膵臓FoundationのNCIの胞子によってサポートされていました。

B7-H1/PD-L1、免疫調節細胞表面タンパク質のB7ファミリーのメンバーは、プログラム死1（PD、その受容体との相互作用を介して細胞性免疫応答の負の調節に重要な役割を果たしている-1）1,2。腫瘍細胞によるB7-H1の過剰発現は、黒色腫、神経膠芽腫、肺の癌、乳癌、大腸癌、卵巣癌、腎細胞を含むヒトの癌の数で注目されており、抗腫瘍損なうことが示されているTシャツを細胞免疫3-8。 最近では、膵臓腺癌組織によるB7-H1の発現は、潜在的な予後マーカー9,10として同定されている。さらに、膵臓癌のマウスモデルにおいて、B7-H1の封鎖は、抗腫瘍応答11を生成することが示されている。これらのデータは、潜在的な治療標的としてのB7-H1の重要性を示唆している。抗B7-H1の封鎖抗体は、したがって、複数の人間のための臨床試験で検証されています黒色腫と肺、結腸、腎臓、胃、膵臓12の癌を含む固形腫瘍。 最終的にはB7-H1標的療法の恩恵を受ける患者を識別できるようにするためには、B7-H1およびB7-H1の封鎖抗体による処置に対する患者の応答の発現と局在の相関関係を調査することが重要です。免疫組織化学を通してヒト膵臓腺癌組織におけるB7-H1の発現を調べると、この共抑制シグナル伝達分子は、腫瘍の微小環境における腫瘍免疫の抑制に寄与する方法のより良い理解を与える。チェンと同僚によって開発された抗B7-H1モノクローナル抗体（クローン5H1）は、ヒト腫瘍組織の複数のタイプ4,8の凍結切片で信頼性の高い染色結果を生成することが示されているが、パラフィン包埋されたスライド上の染色がするまで挑戦していた最近13から18。我々は、B7-H1 stainiを開発しました膵臓腺癌組織のパラフィン包埋されたスライドのngのプロトコル。ここで説明するB7-H1染色プロトコールは少し背景に、B7-H1の一貫性のある膜と細胞質染色を生成します。 1。デparafinization <ol><li>焼く20分間55℃でスライドします。 </li><li> 10分間、キシレン中の化学フード内、浸すスライド。 </li><li> 10分間、新鮮なキシレンに浸してスライドする。 </li><li>浸し、5分間100％エタノール中でスライドします。 </li><li> 5分間、新鮮な100％エタノールでスライドを浸す。 </li><li>浸し、5分間、95％エタノールにスライド。 </li><li>浸し、5分間、80％エタノールにスライド。 </li><li> 5分間脱イオン水でスライドを浸す。 </li></ol> 2。抗原回復<ol><li>その後125℃で30秒間のTris-EDTA緩衝液（pH9.0）と圧力鍋の熱、90℃で10秒間、Cに浸しスライド。 </li><li> 60分間のクールなスライドしてみましょう。 </li><li> 5分間TBSTに浸しスライド; REPE2倍で。 </li></ol> 3。染色<ol><li>スライド上の組織の周りのソリューションを拭き取ってください。疎水性のペンで組織の周りに円をマークします。 </li><li>湿度室でスライドを置き、全体の染色プロセス全体を通して、スライド上の組織の乾燥を避ける。 5分間ペルオキシダーゼブロック内のスライドとインキュベートスライド上に疎水性の円内にペルオキシダーゼブロックの代わりに1滴（完全に組織領域をカバーする約200μl以上）。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。 </li><li> 15分間インキュベートし、各スライドにブロッキングバッファーをブロックエースの代わりに一滴。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。 </li><li> 15分間インキュベートし、各スライド上のアビジン溶液の代わりに一滴。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。 </li><li> 15分間インキュベートし、各スライドにビオチン溶液の代わりに一滴。 </li><li> SLIすすぎTBSTでデ、その後5分間、TBSTで配置します。 </li><li> 4で200μlのTBST中κアイソタイプコントロールマウス抗B7-H1モノクローナル抗体（クローン5H1）またはマウスIgG1の1:1000希釈でスライドをインキュベート℃で22時間+ / - のC一晩 - 1時間。 TBST中で最終的な抗体濃度が2.57μg/ mLである。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。二回繰り返します。 </li><li> 30分間1:500濃度（200μlの2％ヤギ血清溶液中）でビオチン結合ラット抗マウスIgG1二次抗体を用いてスライドをインキュベートする。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。二回繰り返します。 </li><li> 15分間インキュベートし、各スライド上のストレプトアビジン - ビオチン複合体の場所は1滴（使用前に少なくとも30分を準備しなければなりません）。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。二回繰り返します。 </li><li> 4分間インキュベートし、各スライド上の増幅試薬の代わりに1滴（TBSTで2:1に希釈したもの）。 </li><li> TとスライドをすすぐBSTは、その後5分間TBSTで配置します。二回繰り返します。 </li><li> 15分間インキュベートし、各スライド上のストレプトアビジン-H​​RPの代わりに一滴。 </li><li> 5分間、TBSTで開催その後、TBSTでスライドを洗浄します。二回繰り返します。 </li><li>場所は1つ各スライドの3,3 - ジアミノベンジジン（DAB）基板とクロモゲンのドロップと2分間発色する。 </li><li>すぐに発色した後、1〜2分間ddH 2 Oでスライドを洗浄します。 </li><li> 90秒のヘマトキシリン​​1滴でスライドをインキュベートする。 </li><li> 1〜2分間ddH 2 Oで洗浄します。 </li><li> 1〜2分間石鹸水道水で洗ってください。 </li><li> 1〜2分間ddH 2 Oで洗浄します。 </li></ol> 4。脱水および取り付け<ol><li> 3分間80％エタノール中でスライドを浸す。 </li><li> 3分間95％エタノール中でスライドを浸す。 </li><li> 5分間100％エタノールにスライドを浸す。繰り返します。 </li><li> 5分間キシレンにスライドを浸す。繰り返します。 </li><li>一滴を追加スライドとカバースリップ場所にCytoseal 60。 </li></ol> 表1は、上記の手順で使用する試薬。

<ol>
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凍結切片上のB7-H1の免疫組織化学的染色は、癌3,4の様々な先行研究で報告されている。しかし、臨床腫瘍サンプルは、通常、パラフィン包埋ブロックの形で提供されています。 5H1モノクローナル抗体は、より最近に正常腎細胞癌、子宮頚癌、膀胱癌、悪性黒色腫13から18を含むパラフィン包埋腫瘍組織の染色に応用されてきた。パラフィン包埋9,19または他のモノクロ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> CSAシステム</td><td>ダコ</td><td> K1500 </td><td>ペルオキシダーゼブロック、ストレプトアビジン - ビオチン複合、増幅試薬、ストレプトアビジン-H​​RPおよびDAB基質とクロモゲン：プロトコルに記載されている以下の試薬が含まれています</td></tr><tr><td>アビジン/ビオチンブロッキングキット</td><td>ベクトル</td><td> SP-2001 </td><td></td></tr><tr><td>ブロックエース</td><td> Serotec社</td><td> BUF029 </td><td></td></tr><tr><td>ビオチン抗マウスIgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td>マウスIgG1 Kアイソタイプコントロール</td><td> eBioscience社</td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS（トリス緩衝生理食塩水）、10X </td><td> Cellgro </td><td> 46から012-CM </td><td> 10xTBSは80g / LのNaClおよび24.2 g / LのTIRを含んでいます。 洗浄用ddH2Oで1Xに希釈; </td></tr><tr><td>ツイーン20 </td><td>シグマアルドリッチ</td><td> P1379 </td><td> 1ミリリットルはTBSTを作るために1X TBSの1Lに加え</td></tr><tr><td>ターゲット検索、20X </td><td> Celerusウェーブ</td><td> 014-3000-000 </td><td> ddH 2 Oで1倍に希釈し</td></tr><tr><td> 190プルーフエチルアルコール</td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> 200プルーフエチルアルコール</td><td> Pharmo-Aaper </td><td> 111000200 </td><td></td></tr><tr><td>キシレン</td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td>ヘマトキシリン</td><td>リチャード·アラン·サイエンティフィック</td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td>リチャード·アラン·サイエンティフィック</td><td> 8310から4 </td><td></td></tr><tr><td>カバースリップ</td><td>コー​​ニング</td><td> 2940-245 </td><td></td></tr><tr><td>湿度室</td><td>シグマ</td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1試薬と機器。 </td></tr></tbody></table>

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

B7-H1/PD-L1、免疫調節細胞表面タンパク質のB7ファミリーのメンバーは、プログラム死1（PD、その受容体との相互作用を介して細胞性免疫応答の負の調節に重要な役割を果たしている-1）1,2。腫瘍細胞によるB7-H1の過剰発現は、黒色腫、神経膠芽腫、肺の癌、乳癌、大腸癌、卵巣癌、腎細胞を含むヒトの癌の数で注目されており、抗腫瘍損なうことが示されているTシャツを細胞免疫3-8。 最近では、膵臓腺癌組織によるB7-H1の発現は、潜在的な予後マーカー9,10として同定されている。さらに、膵臓癌のマウスモデルにおいて、B7-H1の封鎖は、抗腫瘍応答11を生成することが示されている。これらのデータは、潜在的な治療標的としてのB7-H1の重要性を示唆している。抗B7-H1の封鎖抗体が故にmulのための臨床試験で検証されています黒色腫と肺、結腸、腎臓、胃、膵臓癌を含む12のtipleヒト固形腫瘍。 最終的にはB7-H1標的療法の恩恵を受ける患者を識別できるようにするためには、B7-H1およびB7-H1の封鎖抗体による処置に対する患者の応答の発現と局在の相関関係を調査することが重要です。免疫組織化学を通してヒト膵臓腺癌組織におけるB7-H1の発現を調べると、この共抑制シグナル伝達分子は、腫瘍の微小環境における腫瘍免疫の抑制に寄与する方法のより良い理解を与える。チェンと同僚によって開発された抗B7-H1モノクローナル抗体（クローン5H1）は、ヒト腫瘍組織の複数のタイプ4,8の凍結切片で信頼性の高い染色結果を生成することが示されているが、パラフィン包埋されたスライド上の染色がするまで挑戦していた最近13から18。我々は、devがある膵臓腺癌組織のパラフィン包埋されたスライドの駆け落ちB7-H1染色プロトコール。ここで説明するB7-H1染色プロトコールは少し背景に、B7-H1の一貫性のある膜と細胞質染色を生成します。

B7-H1の成功した免疫組織化学的染色は、膵臓腺癌におけるB7-H1（茶色信号のDAB発色）の異種発現を実証します。他の人がB7-H1またはB7-H1（ 図2、図3）を少し表現のいずれかの主に細胞質発現を持っているのに対し、一部の膵臓腺癌細胞がB7-H1の主に膜状の発現（ 図1A）を持っています。通常の膵管上皮は少しB7-H1（ 図4）を発現する...

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）とPD-1への結合は、腫瘍の微小環境における主要な腫瘍誘発免疫抑制信号を提供します。膵臓腺癌におけるB7-H1の発現と局在を特徴付けるための免疫組織化学的染色法はここで説明されます。

膵臓腺癌組織のパラフィン包埋したスライド上のB7-H1（PD-L1）の免疫組織化学的染色

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 ビュー.  The Johns Hopkins University School of Medicine. B7-H1（PD-L1）とPD-1への結合は、腫瘍の微小環境における主要な腫瘍誘発免疫抑制信号を提供します。膵臓腺癌におけるB7-H1の発現と局在を特徴付けるための免疫組織化学的染色法はここで説明されます。

ビデオ: 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.

ヒト膵島の染色プロトコル

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

医学

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.

齧歯尿生殖洞間充織とヒト幹細胞の組織の組換えを用いたヒト前立腺上皮の形成

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.

膵臓癌の進行の生物発光同所性モデル

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.

経皮針生検によって得られた骨格筋組織から単離されたミトコンドリアの調製と呼吸計測評価

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.

ロボット中央膵の吻合をエンド ツー エンドのアプリケーション

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.

マウス モデルにおける膵島の門脈内移植

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.

パラフィン埋め込み組織におけるヒトおよびマウス好中球外細胞トラップの免疫蛍光標識

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.

膵臓頭部における膵内インスリン腫のロボット的発風

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.

バソフェナントロリンベースの比色アッセイを用いた組織非ヘム鉄含有量の測定

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.