EB, KMB, ve HX eşit katkıda bulunurlar. Bu çalışmada, gastrointestinal kanserler P50 CA062924, NIH K23 CA148964, Lustgarten Vakfı, Viragh Vakfı, ASCO Genç Araştırmacı Ödülü, NIH 5T32 CA0090701-28 Eğitim Bursu, Sol Goldman Pankreas Kanseri Merkezi ve Ulusal Pankreas Vakfı NCI SPORE tarafından desteklenmiştir.

B7-H1/PD-L1, bağışıklık düzenleyici bir hücre yüzey proteinlerinin B7 ailesinin bir üyesi, (PD programlanmış ölüm-1, reseptörü ile etkileşim yoluyla hücre-aracılı bağışıklık tepkilerini negatif düzenlenmesi önemli bir rol oynar -1) 1,2. Tümör hücreleri tarafından B7-H1 aşırı ekspresyonu melanom, glioblastom, ve akciğer, meme, kolon, yumurtalık, böbrek ve hücre karsinomları da dahil olmak üzere insan kanserleri, bir dizi belirtildiği gibi, ve anti-tümör zarar gösterilmiştir T- Hücre bağışıklık 3-8. Son zamanlarda, pankreatik adenokarsinoma dokular tarafından B7-H1 ekspresyonu potansiyel bir prognostik belirteçtir 9,10 olarak tespit edilmiştir. Buna ek olarak, pankreas kanseri, bir fare modelinde, B7-H1 blokajı, bir anti-tümör cevabı 11 üretmek için gösterilmiştir. Bu veriler potansiyel bir terapötik hedef olarak B7-H1 önemini göstermektedir. Anti-B7-H1 blokajı antikorlar nedenle birden çok insan için klinik deneylerle test ediliyormelanom ve akciğer, kolon, böbrek, mide ve pankreas 12 kanserleri dahil olmak üzere solid tümörler. Sonunda tedaviler hedefleme B7-H1 yararlanacak hastaların tespit edebilmek için, bu ifade ve B7-H1 ve B7-H1 blokajı antikorlar ile hastanın tedaviye yanıtı lokalizasyonu arasındaki korelasyonu araştırmak için kritik öneme sahiptir. Immünhistokimya ile insan pankreas adenokarsinomu dokularında B7-H1 ekspresyonu incelenmesi bu co-inhibitör sinyal molekülü tümör mikroçevresindeki antitümör bağışıklık baskılanması nasıl katkıda bulunduğunu daha iyi bir anlayış verecektir. Chen ve arkadaşları tarafından geliştirilen anti-B7-H1 monoklonal antikor (klon 5H1) insan neoplastik doku 4,8 çok tip cryosections güvenilir boyama sonuçları üretmek için gösterilmiştir, ancak parafine gömülü slaytlar leke kadar zor olmuştu Son zamanlarda 13-18. Biz, B7-H1 staini geliştirdikpankreatik adenokarsinoma dokuların parafine gömülü slaytlar için ng protokolü. Burada anlatılan B7-H1 boyama protokolü biraz arka plan ile B7-H1 tutarlı membranöz ve sitoplazmik boyanma üretir. 1. De-parafinization <ol><li> Fırınlama 20 dakika süreyle 55 ° C&#39;de kayar. </li><li> 10 dakika için ksilen içinde bir kimyasal Hood, batırmak kayar. </li><li> 10 dakika için taze ksilen içinde Immerse kayar. </li><li> Daldırın 5 dakika için% 100 etanol içinde kayar. </li><li> Daldırın 5 dk için taze% 100 etanol içinde kayar. </li><li> Daldırın 5 dakika için% 95 etanol içinde kayar. </li><li> Daldırın 5 dakika için% 80 etanol içinde kayar. </li><li> Daldırın 5 dk için deiyonize su içinde kayar. </li></ol> 2. Antigen Retrieval <ol><li> Daha sonra 125 ° C&#39;de 30 saniye için Tris-EDTA tampon maddesi (pH 9.0) ve bir düdüklü tencere içinde sıcaklık, 90 ° C 10 sn için sokmak kayar. </li><li> 60 dakika için slaytlar soğumasını bekleyin. </li><li> 5 dakika TBST yılında batırın slaytlar; repeiki kez. </li></ol> 3. Lekelenme <ol><li> Slaytlarda dokusunun etrafında çözümler siliniz. Hidrofobik bir kalem ile doku etrafında bir çember işaretleyin. </li><li> Bir nem odasında slaytlar yerleştirin ve tüm boyama işlemi boyunca slayt üzerinde doku kurumayı önlemek. 5 dakika peroksidaz blok slayt ve inkübe slaytlar üzerinde hidrofobik daire içinde peroksidaz blok Sıra bir damla (tamamen doku alanı kapsayacak şekilde yaklaşık 200 ul veya daha fazla). </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. </li><li> BLOK ACE Yeri bir damla 15 dakika boyunca her slayt ve inkübe tampon engelleme. </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. </li><li> 15 dakika boyunca her slayt ve inkübe üzerinde Avidin çözüm yeri bir damla. </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. </li><li> 15 dakika boyunca her slayt ve inkübe üzerinde Biotin çözüm yeri bir damla. </li><li> Sli durulayınTBST ile des, 5 dakika TBST sonra yer. </li><li> Fare anti-B7-H1 monoklonal antikor seyreltme 1:1,000 (klon 5H1) ya da bir fare IgG1 ile slaytlar inkübe 4 de 200 ul TBST içinde κ izotip kontrol ° C&#39;de 22 saat + / için bir gecede - 1 saat. TBST içinde son antikor konsantrasyonu 2.57 ug / ml &#39;dir. </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. Iki kez tekrarlayın. </li><li> 30 dakika boyunca bir 1:500 yoğunlukta bir biyotin konjuge sıçan anti-fare IgG1 ikincil antikor (200 ul% 2 keçi serumu içinde çözelti) ile slaytlar inkübe edin. </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. Iki kez tekrarlayın. </li><li> Streptavidin-Biotin Complex Sıra bir damla 15 dakika boyunca her slayt ve inkübe üzerinde (kullanımdan önce en az 30 dakika hazırlıklı olmalıdır). </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. Iki kez tekrarlayın. </li><li> 4 dakika için her slayt ve inkübe üzerinde Amplifikasyon Reaktif Sıra bir damla (TBST ile 02:01 seyreltilmiş). </li><li> T ile slaytlar durulayınBST, 5 dk TBST sonra yer. Iki kez tekrarlayın. </li><li> 15 dakika boyunca her slayt ve inkübe üzerinde Streptavidin-HRP Yeri bir damla. </li><li> 5 dk için TBST içinde yer sonra, slaytlar TBST ile yıkayın. Iki kez tekrarlayın. </li><li> Her bir slayt üzerinde bir 3,3-diaminobenzidin damla (DAB) substrat ve kromojen koyun ve 2 dk için renk geliştirmek. </li><li> Hemen renk geliştirme sonra, 1-2 dakika GKD 2 O ile slaytlar yıkayın. </li><li> 90 sn için hematoksilen bir damla ile slaytlar inkübe edin. </li><li> 1-2 dk GKD 2 O ile yıkayın. </li><li> 1-2 dakika sabunlu musluk suyu ile yıkayın. </li><li> 1-2 dk GKD 2 O ile yıkayın. </li></ol> 4. Dehidrasyon ve Montaj <ol><li> 3 dakika için% 80 etanol içinde slaytlar bırakın. </li><li> 3 dakika boyunca% 95 etanol içinde slaytları daldırın. </li><li> 5 dakika için% 100 etanol içinde slaytlar bırakın. Tekrarlayın. </li><li> 5 dk için ksilen içinde slaytlar bırakın. Tekrarlayın. </li><li> Bir damla eklemeSlayt ve yer lamel Cytoseal 60. </li></ol> Tablo 1&#39;de, yukarıda anlatılan prosedürlerde kullanılan reaktifler listelenmiştir.

<ol>
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Çıkar çatışması ilan etti.

Cryosections B7-H1 immünohistokimyasal boyama Kanseri 3,4 çeşitli önceki çalışmalarda rapor edilmiştir. Bununla birlikte, klinik tümör örnekleri genellikle sadece parafine gömülmüş blok şeklinde kullanılabilir. 5H1 monoklonal antikor daha yakın başarıyla renal hücreli karsinom, serviks kanseri, mesane kanseri ve melanom 13-18 gibi parafin tümör dokularının boyama uygulanmıştır. Parafine gömülü 9,19 veya kriyostat diğer monoklonal antikorlar ile pankreatik ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> CSA Sistemi </td><td> Dako </td><td> K1500 </td><td> Peroksidaz blok, streptavidin-biotin kompleksi, amplifikasyon reaktifi, streptavidin-HRP ve DAB substrat ve kromojen: Aşağıdaki protokolde belirtilen reaktifler içerir </td></tr><tr><td> Avidin / Biotin Engelleme Kiti </td><td> Vektör </td><td> SP-2001 </td><td></td></tr><tr><td> Blok ACE </td><td> Serotec </td><td> BUF029 </td><td></td></tr><tr><td> Biotin anti-fare IgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> Fare IgG1 K izotip kontrol </td><td> eBioscience </td><td> 14-4714-85 </td><td></td></tr><tr><td> 10X TBS (salin Tris tamponlu), </td><td> Cellgro </td><td> 46-012-CM </td><td> 10xTBS 80 g / L NaCl ve 24.2 g / L tırlar içerir; Yıkama için ddH2O ile 1X seyreltilir; </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P1379 </td><td> 1 ml TBST yapmak için 1X TBS 1 L eklendi </td></tr><tr><td> Hedef Alma, 20x </td><td> Celerus Dalga </td><td> 014-3000-000 </td><td> GKD 2 O ile 1x Seyreltilmiş </td></tr><tr><td> 190 Proof Etil Alkol </td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> 200 Proof Etil Alkol </td><td> PHARMO-Aaper </td><td> 111000200 </td><td></td></tr><tr><td> Ksilen </td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td> Hematoksilen </td><td> Richard-Allan Bilimsel </td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Richard-Allan Bilimsel </td><td> 8310-4 </td><td></td></tr><tr><td> Lameller </td><td> Corning </td><td> 2940-245 </td><td></td></tr><tr><td> Nem odası </td><td> Sigma </td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tablo 1. Reaktifler ve cihazlar. </td></tr></tbody></table>

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

B7-H1/PD-L1, bağışıklık düzenleyici bir hücre yüzey proteinlerinin B7 ailesinin bir üyesi, (PD programlanmış ölüm-1, reseptörü ile etkileşim yoluyla hücre-aracılı bağışıklık tepkilerini negatif düzenlenmesi önemli bir rol oynar -1) 1,2. Tümör hücreleri tarafından B7-H1 aşırı ekspresyonu melanom, glioblastom, ve akciğer, meme, kolon, yumurtalık, böbrek ve hücre karsinomları da dahil olmak üzere insan kanserleri, bir dizi belirtildiği gibi, ve anti-tümör zarar gösterilmiştir T- Hücre bağışıklık 3-8. Son zamanlarda, pankreatik adenokarsinoma dokular tarafından B7-H1 ekspresyonu potansiyel bir prognostik belirteçtir 9,10 olarak tespit edilmiştir. Buna ek olarak, pankreas kanseri, bir fare modelinde, B7-H1 blokajı, bir anti-tümör cevabı 11 üretmek için gösterilmiştir. Bu veriler potansiyel bir terapötik hedef olarak B7-H1 önemini göstermektedir. Anti-B7-H1 blokajı antikorlar nedenle mul için klinik deneylerle test ediliyormelanom ve akciğer, kolon, böbrek, mide ve pankreas kanserleri de dahil olmak üzere 12 tiplemeleriyle insan solid tümörler. Sonunda tedaviler hedefleme B7-H1 yararlanacak hastaların tespit edebilmek için, bu ifade ve B7-H1 ve B7-H1 blokajı antikorlar ile hastanın tedaviye yanıtı lokalizasyonu arasındaki korelasyonu araştırmak için kritik öneme sahiptir. Immünhistokimya ile insan pankreas adenokarsinomu dokularında B7-H1 ekspresyonu incelenmesi bu co-inhibitör sinyal molekülü tümör mikroçevresindeki antitümör bağışıklık baskılanması nasıl katkıda bulunduğunu daha iyi bir anlayış verecektir. Chen ve arkadaşları tarafından geliştirilen anti-B7-H1 monoklonal antikor (klon 5H1) insan neoplastik doku 4,8 çok tip cryosections güvenilir boyama sonuçları üretmek için gösterilmiştir, ancak parafine gömülü slaytlar leke kadar zor olmuştu Son zamanlarda 13-18. Biz dev varpankreatik adenokarsinoma dokuların parafine gömülü slaytlar için kaçtı B7-H1 boyama protokolü. Burada anlatılan B7-H1 boyama protokolü biraz arka plan ile B7-H1 tutarlı membranöz ve sitoplazmik boyanma üretir.

B7-H1 başarılı immunhistokimyasal pankreatik adenokarsinoma B7-H1 (kahverengi işaretleri DAB renk geliştirilmesi) heterojen ifadesi gösterecektir. Diğerleri B7-H1 veya B7-H1 (Şekil 2 ve 3) az ifade ya ağırlıklı sitoplazmik ifade var ancak bazı pankreatik adenokarsinoma hücreleri B7-H1 ağırlıklı membranöz ekspresyonu (Şekil 1A) var. Normal pankreatik kanal epitel (Şekil 4) B7-H1 biraz ifade eder. Bir fare IgG1 κ izotip kontrol antikor ile lekelenmes...

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) ve PD-1 olarak bağlayıcı tümörün mikroçevresindeki önemli bir tümör kaynaklı immünsüpresif sinyal verin. Ifade ve pankreatik adenokarsinoma B7-H1 lokalizasyonu karakterize etmek için immünohistokimyasal boyama tekniği burada tanımlanmıştır.

Pankreas Adenokarsinom Doku B7-H1 immünohistokimyasal boyanma (PD-L1) parafin Slaytlar

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 Görüntüleme.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) ve PD-1 olarak bağlayıcı tümörün mikroçevresindeki önemli bir tümör kaynaklı immünsüpresif sinyal verin. Ifade ve pankreatik adenokarsinoma B7-H1 lokalizasyonu karakterize etmek için immünohistokimyasal boyama tekniği burada tanımlanmıştır.

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.

İnsan Pankreas Adacıklar için boyama Protokoller

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

Tıp

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.

Kemirgen Ürogenital Sinüs mezenşimin ve İnsan Kök Hücre Doku Rekombinasyon kullanarak İnsan Prostat epiteli oluşumu

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

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.

Perkütan İğne Biyopsisi ile Elde Edilen İskelet Kası Doku İzole Mitokondri hazırlanması ve Respirometrik Değerlendirmesi

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.

Uçtan uca anastomoz Robotik Merkezi Pancreatectomy içinde uygulanması

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

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.

Parafin-Gömülü Dokuda İnsan ve Mürin Nötrofil Ekstrasellüler Tuzaklarının İmmünofloresan Etiketlemesi

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.

Pankreas Kafada Pankreas İçi İnülinomanın Robotik Enükleasyonu

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.

Batofenantropin Bazlı Kolorimetrik Tahlil Kullanılarak Doku Non-Heme Demir İçeriğinin Ölçülmesi

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

Tissue Embedding and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Murin Pulmoner Venlerde Miyokard Kılıflarının Doku Gömülmesi ve İmmünofloresan Boyanması

<meta charset="utf8"></head><body>Kemirgen Gözbebeklerinde Retina Kalınlığı ve Ganglion Hücre Tabakası Sayılarının Değerlendirilmesi

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

<meta charset="utf8"></head><body>Retinayı değerlendirmek için sıçan ve fare gözbebeklerinin histolojik slaytlarının hazırlanması ve analizi

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

Retinayı değerlendirmek için sıçan ve fare gözbebeklerinin histolojik slaytlarının hazırlanması ve analizi

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...