Os autores não têm reconhecimento.

Todos os procedimentos experimentais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade Metropolitana de Tóquio (IACUC). Os animais foram alojados e manuseados de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório e o Guia para o Cuidado e Uso de Animais Experimentais do Conselho Canadense de Cuidados com Animais. Utilizamos animais criados e mantidos no Laboratório de Morfologia Funcional, Departamento de Ciências da Saúde da Fronteira, Universidade Metropolitana de Tóquio, Japão. 1. Cuidados com animais e habitação <ol><li> Obtenha 7 S. murinus (3 fêmeas e 4 machos pesando 70-90 g) de uma colônia de criação fechada. </li><li> Cale o S. murinus individualmente após o desmame (20 d após o nascimento ) em gaiolas de plástico equipadas com uma caixa de ninho de madeira contendo tiras de papel. Mantenha-os em uma sala de animais convencionalmente condicionada: 23-27 ° C, sem controle de umidade e uma luz de 12:12 h: ciclo escuro. Supply commercPellets de trutas e água ad libitum. </li></ol> 2. Preparação de Tecidos <ol><li> Para preparar 4% (p / v) de paraformaldeído (PFA), dissolver 40 g de PFA em 1000 mL de solução salina tamponada com fosfato 0,01 M (PBS, pH 7,4). Em uma aspiradora, misture usando um vórtice até a solução ficar limpa. Armazene 4% de PFA O / N a 4 ° C. Cuidado: Use equipamento de proteção pessoal apropriado (PPE) ao manusear o PFA. </li><li> Para perfurar e consertar o animal, anestesiar com éter e depois dar-lhe uma injeção intraperitoneal de somnopentilo (pentobarbital sódico, 0,6 mL / kg de peso corporal). </li><li> Depois que o animal é completamente narcotizado, abra a cavidade abdominal com um bisturi, criando uma incisão abdominal na linha média com 3 cm de comprimento. Ao incitar a veia cava inferior para exsanguinating, insira um catéter retrogradável na aorta abdominal no nível imediatamente acima da bifurcação desta artéria nas artérias ilíacas comuns. </li><li> Injetar somnopentil (pentobarbital assimDium, 1,0 mL / kg de peso corporal). Após a eutanásia completa, perfusar com PBS 0,01 M (pH 7,4) e depois com 4% de PFA numa aspiradora. </li><li> Após a perfusão, injete 2-3 mL de látex branco de neoprene (diluição do látex de neoprene branco 3: 2 com água destilada (DW)) para rotular os vasos sanguíneos. </li><li> Extraia os órgãos abdominais, incluindo o fígado, a vesícula biliar, o ducto biliar comum, o duodeno e o pâncreas em bloco, com os nervos e vasos relacionados. </li><li> Injetar aproximadamente 0,1 mL de látex azul de neoprene (adicionar uma gota de tinta azul a 10 mL do látex de neoprene branco diluído) até a vesícula biliar para rotular o sistema biliar. </li><li> Pós-fixação durante a noite com 4% de PFA a 4 ° C para imunomarcação total. Atenção: o PFA é tóxico. Evite manipular diretamente o PFA. O PFA deve ser usado em uma chaminé. </li></ol> 3. Imunohistoquímica de montagem total Nota: Ao longo do protocolo, incluindo durante a lavagem, incubação de anticorpos e coloração,O espécime deve permanecer no nutator, balançando suavemente à TA ou a 4 ° C. <ol><li> Dia 1: Tratamento enzimático e inativação da potencial peroxidase endógena <ol><li> Lave o espécime preparado com DW 4x por 1 h cada à temperatura ambiente em um frasco de vidro de tamanho apropriado. Balance o espécime gentilmente sobre o nutator para remover o PFA. Evite danificar o espécime ao trocar soluções. </li><li> Para preparar 1% (p / v) de ácido ortoperiodico, dissolva 1 g de ácido ortoperiodico em 100 mL de DW. </li><li> Incubar a amostra em 1% de ácido ortoperiodico durante 20 minutos à TA para evitar qualquer reação intrínseca à peroxidase. </li><li> Prepare 0,004% (p / v) de papaína por dissolução de 0,004 g de papaína em 100 mL de tampão Tris-HCl 0,025 mol / L (pH 7,6). </li><li> Lave o espécime com DW por 10 minutos à TA. </li><li> Incubar o espécime em 0,004% de papaia recém-preparada durante 2 h a 37 ° C em banho a temperatura constante com balanço suave. </li><li> Lave o espécime com DW por 50-60 minutos na RT. </li><li> Armazene o espécime em 4% de PFA a 4 ° CO / N. </li></ol></li><li> Dia 2: tratamento enzimático <ol><li> Lave o espécime armazenado com DW 4 vezes durante 1 h cada à TA. </li><li> Incubar o espécime em 0,004% de papaia recém-preparada durante 2 h a 37 ° C em banho a temperatura constante com balanço suave. </li><li> Lave o espécime com DW durante 50-60 minutos à RT. Armazene o espécime em 4% de PFA a 4 ° CO / N. </li></ol></li><li> Dia 3: tratamento de congelação <ol><li> Lave o espécime armazenado com DW 4x durante 1 h cada à temperatura ambiente, como acima. </li><li> Preparar 2,5% (p / v), 5% (p / v) e 10% (p / v) de sacarose dissolvendo 2,5 g, 5 g e 10 g de sacarose, respectivamente, em 100 mL de PBS 0,01 M ( PH 7,4). </li><li> Mergulhe o espécime em 2,5% (p / v), 5% (p / v) e 10% (p / v) de sacarose por 30 min cada. </li><li> Congele o espécime a -20 ° C durante 30 a 60 min e depois descongelá-lo completamente à TA; Repita o ciclo 3x. </li><li> TopoRepare 2% de Triton X-100 (v / v), adicione 2 mL de Triton X-100 a 100 mL de PBS 0,01 M (pH 7,4). </li><li> Armazene o espécime em 2% de Triton X-100 a 4 ° CO / N. </li></ol></li><li> Dia 4: incubação primária de anticorpos <ol><li> Prepare a solução de diluição do anticorpo primário dissolvendo 0,2 g de albumina de soro bovino (BSA), 0,3 mL de Triton X-100 e 0,1 g de azida de sódio em 100 mL de PBS 0,01 M (pH 7,4). Atenção: A azida de sódio é extremamente tóxica. A inalação e o toque devem ser evitados. Use PPE apropriado. </li><li> Diluir o anticorpo primário (NFP) 1: 600 no tampão de diluição acima (passo 3.4.1). Incube o espécime com anticorpo NFP a 4 ° C por 3-6 dias, mantendo o espécime balançando suavemente no nutator. Espécimes maiores exigirão tempos de incubação aumentados. </li></ol></li><li> Incubação secundária de anticorpos <ol><li> Lave o espécime em PBS 4x durante 1 h cada à TA para remover o anticorpo primário não ligado. </lI&gt; <li> Preparar a solução de diluição para o anticorpo secundário dissolvendo 0,2 g de BSA e 0,3 mL de Triton X-100 em 100 mL de PBS 0,01 M (pH 7,4). </li><li> Diluir o anticorpo secundário (IgG-HRP anti-rato anti-rato purificado por afinidade conjugado com peroxidase) 1: 600 no tampão de diluição (passo 3.5.2). Incube o espécime com o anticorpo secundário a 4 ° C durante 3 dias e mantenha o espécime balançando suavemente na porca. </li></ol></li><li> Coloração <ol><li> Prepare a solução de coloração DAB adicionando 0,002 g de tetrahidrocloreto de 3,3&#39;-diaminobenzidina (DAB) a 100 mL de tampão Tris-HCl 0,05 mol / L (pH 7,6) sob uma aspiradora, mantendo a solução no escuro. </li><li> Lave os espécimes à RT em PBS 4x por 1 h cada. </li><li> Adicione 10 μL de 30% de H 2 O 2 em solução de coloração DAB recém-preparada e incuba com a solução a 4 ° CO / N ou durante 3 dias enquanto se meca suavemente no nutator. </li><li> Pare a reação por plaCing o espécime em PBS com 0,04% de azida de sódio quando a intensidade de coloração ideal é atingida. NOTA: Evite o uso de azida de sódio até este passo, pois inibe a peroxidase de rábano. </li></ol></li><li> Imaginando o tecido manchado <ol><li> Transfira cuidadosamente o espécime para um prato de vidro petri (5 cm de altura, 10 cm de diâmetro) contendo PBS. Capture imagens de montagem total usando uma câmera montada em um estereomicroscópio. NOTA: O tecido manchado de montagem total deve ser fotografado enquanto mergulhou completamente em PBS em um prato de vidro transparente. </li></ol></li></ol>

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Os autores não declaram conflitos de interesse.

Este trabalho descreve o procedimento experimental para a visualização da inervação do trato biliar extra-hepático usando um anticorpo contra a proteína do neurofilamento. Este protocolo foi adaptado do protocolo descrito por Kuratani e Tanaka 1 , 2 , 3 . Para esta experiência, planeje a linha de tempo para cada uma das experiências, já que a abordagem de coloração total continua por 2-3 ...

A muscaria Musk Muscus , Suncus murinus , pertence à ordem Insectivora e Soricidae familiar. Este pequeno mamífero é distribuído em todo o Sudeste Asiático e habita casas e áreas gramadas que se situam perto de habitações humanas ou canetas de gado 4 . Esta espécie exibe características morfológicas gerais mais semelhantes aos humanos do que outros animais de laboratório, como o mouse, o rato e o coelho 5 . Anteriormente, foi utilizada imunomarquência total com um marcador de neurônio periférico para a proteína de neurofilamento de S. murinus (NFP) para rotular os nervos periféricos no pâncreas 6 , a papila duodenal principal 7 , o píloro 8 , a vesícula biliar 5 e o trato biliar extra-hepático 9 . NFP é um complexo macromolecular que faz parte do citoesqueleto neuronal maduro. Proteína relacionada ao neurofilamentoMede as interações entre NFP e o zygosoma. Tanto a função enzimática quanto a estrutura das proteínas de ligação são reguladas pelo NFP 10 . O complexo NFP é composto de três polipéptidos: NF-L (70 kDa), NF-M (150-160 kDa) e NF-H (200 kDa). O NFP pode ser encontrado em axônios neuronais no sistema nervoso central e periférico. O anticorpo anti-NFP humano mostrou rotular os axônios do sistema nervoso central e periférico. Investigações anatômicas e eletrofisiológicas demonstraram que o esfíncter, a vesícula biliar e o trato gastrointestinal proximal estão conectados 11 , 12 , 13 , 14 . No entanto, as características morfológicas de suas conexões neuronais ainda não foram definidas. Este trabalho demonstra o uso de um método de coloração imuno-histoquimica de todo o suporte para rotular a inervação do S. murinus bilCom um anticorpo NFP.

<table><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>I-0125</td><td></td></tr></tbody></table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

Este trabalho descreve detalhadamente o método de coloração de imuno-histoquímica total, usando anticorpo de proteína de neurofilamento para rotular a inervação do trato biliar em Suncus murinus ( S. murinus &#160; ). Primeiro, a amostra foi dissecada de S. murinus e fixada em paraformaldeído a 4% (PFA). Em segundo lugar, foram realizados um tratamento enzimático e potencial inativação da peroxidase endógena. O espécime foi então exposto ao anticorpo primário, anticorpo anti-proteína de neurofilamento, durante 3-6 dias. Foi então incubado com o anticorpo secundário conjugado com peroxidase de rábano. A reação de cor foi revelada fazendo reagir o espécime com um substrato de 3,3&#39;-diaminobenzidina (DAB). Este método pode ser aplicado para analisar a inervação de todos os órgãos viscerais. Além disso, este protocolo também pode ser adaptado para testar outros anticorpos neuronais, mas a otimização do antibodiO primeiro deve ser feito primeiro. Este método foi originalmente introduzido por Kuratani e Tanaka 1 , 2 , 3 .

As Figuras 1 a 4 mostram resultados típicos para fibras nervosas NFP positivas no trato biliar extra-hepático em S. murinus . O anticorpo contra NFP reproduzivelmente rotulou a inervação em toda a imagem do trato extra-hepático biliar ( Figura 1 ), vesícula biliar ( Figura 2 ), ducto biliar superior ( Figura 3 ) e área de papila duodenal ( <strong cl...

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Uma abordagem imuno-histoquímica de montagem total, para visualizar a expressão da proteína de neurofilamento no trato biliar extra-hepático em Suncus murinus. É apresentado aqui. Este protocolo pode ser usado para analisar a inervação de todos os órgãos viscerais em S. murinus ou outras espécies.

Usando um método imunohistoquímico de montagem total para estudar a preservação do trato biliar em<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

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Research

JoVE Journal

Biology

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K visualizações.  Tokyo Metropolitan University.  Uma abordagem imuno-histoquímica de montagem total, para visualizar a expressão da proteína de neurofilamento no trato biliar extra-hepático em Suncus murinus. É apresentado aqui. Este protocolo pode ser usado para analisar a inervação de todos os órgãos viscerais em S. murinus ou outras espécies. 

Vídeo: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunostaining para Visualize Murino desenvolvimento do sistema nervoso entérico

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

Biologia do Desenvolvimento

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

In vivo localização de imunofluorescência para avaliação de anticorpo terapêutico e diagnóstico biodistribuição em pesquisa de câncer

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Pesquisa do câncer

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Determinando a densidade do ducto biliar no fígado do rato

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Ligadura do ducto biliar em ratos: Indução de Inflammatory Lesão hepática e fibrose por colestase obstrutiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Medicina

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imagem do Sistema Nervoso Entérico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Neurociência

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Visualização das células intersticiais de Cajal Network (ICC) em ratos

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Biologia

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Imagem latente intravital de linfócitos intraepithelial no intestino pequeno murine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Imunologia e Infecção

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

Um Método de Esclarecimento Novo para Visualize biliar Degeneração Durante fígado metamorfose em lampreia de mar ( Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Murine Precision-Cut Liver Slices como um modelo ex vivo de biologia hepática

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

Localização, Dissecção e Análise do Gânglio Murine Stellate

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

Usando um método imunohistoquímico de montagem total para estudar a preservação do trato biliar em

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