Agradecemos aos nossos colegas em RIKEN-BSI para os seus apoios e conselhos gentilmente frutífera durante este trabalho. Eu gostaria de expressar minha sincera gratidão ao Professor Shigemi Mori, Professor G bor Juh sz, os drs. Kiyoji Matsuyama, Katalin AK kesi e rp d Dobolyi que me aconselhou a estudar o método de microdiálise durante o curso de pós-graduação na Universidade de Pós-Graduação de Estudos Avançados, Japão. Finalmente, agradecemos o Sr. Dr. Chris Sarnecky e Akitsugu Takasu para os seus apoios em gravação de vídeo.

Etapa 1. Preparação de implantação da sonda <ol><li> Anestesia (não mostrado no vídeo) Anestesiar cada ave com uma injeção de um interperitoneal ketamina (12,5 peso corporal mg / kg) ea mistura de xilazina (25 mg / kg) para produzir um nível constante de anestesia cirúrgica. </li><li> Fix uma cabeça a um quadro estereotáxico Use Xylocaine (TM) de geléia para reduzir as dores locais de pressão bares orelha &#39;. A cabeça de pássaro é fixado em um aparelho estereotáxico consistindo de barras de ouvido e um suporte de bico que mantém a cabeça em um ângulo de 45 °. Durante a cirurgia estereotáxica para implantação, aquecer o corpo de ave por pad cirúrgica com controle térmico (temperatura = 34 ~ 36 ° C). </li><li> Desinfectar área cirúrgica Desinfectar o campo cirúrgico com isodine (TM) antes da cirurgia. </li></ol> Etapa 2. Posicionamento estereotáxica <ol><li> Alinhamento ponto de referência Alinhar sonda dummy (vidro micropipeta) para o Lambda (Y-ponto) e marca Y-ponto com a pipeta dye-cheia. Restantes Dextran-TMR do estudo tracer última foi usado no vídeo. </li><li> Incisão da pele Retire a pele e expor a superfície do crânio. Então, limpar a superfície do crânio com cotonetes. </li><li> Posicionamento da cânula dummy (na direção horizontal) Mover a cânula dummy para o site de destino (AP eixo e coordenação Lateral) </li><li> Craniotomia pequena e incisão da dura-máter. Use uma pinça fina para craniotomia e uma agulha de injeção fina (30 G) para incisões da dura-máter. Então, limpar a superfície do crânio. </li><li> Posicionamento final (implantação) da cânula dummy Localize a ponta da cânula guia em 0,30 mm acima da sonda de microdiálise destina-região de destino. Por fim, limpar a superfície do crânio de novo, porque uma superfície limpa e seca é necessária para a ligação difícil. </li></ol> Etapa 3. Pré-tratamento da superfície craniana (O procedimento semelhante para &quot;restauração direta enchimento&quot; do produto dental deve ser empregada.) <ol><li> Tratamento primário <ol><li> Mix líquido PRIMAR A com B em um prato. </li><li> Aplique a mistura sobre a superfície do crânio com uma pequena escova. </li><li> Deixe descansar por 30 segundos. </li><li> Seco com fluxo de ar suave. (Não lavar.) </li></ol></li><li> Aplicação do agente de união <ol><li> Aplicar o agente de união na superfície do crânio com uma pequena escova. </li><li> Aplicar um fluxo de ar suave. (Não lavar.) </li><li> Luz cura por 20 segundos. </li></ol></li></ol> Etapa 4. Montagem de resina composta <ol><li> Montagem de resina com plugger planície Se um plugger Plain não estiver disponível, uma espátula de micro-pode ser usado em seu lugar. </li><li> Fotopolimerização </li><li> Incremental de montagem Incremental de montagem é recomendada para garantir a reação completa da resina. Para a montagem incremental, aplicar agente de união antes de cada montagem. </li><li> Moldar a resina composta e fotopolimerizador para endurecimento final. </li><li> Final da cirurgia </li></ol> Limpar a área cirúrgica, aplicar antibiótico (gentamicina pomada), e remover o animal da chama estereotáxica. Então, manter o animal em uma câmara de termo-controlada (temperatura = 33-35 ° C) para recuperação pós-anestésica. Conforme mostrado no vídeo, nem qualquer comportamento anormal foi observado em nenhum dos animais no dia seguinte após a cirurgia.

<ol>
	<li>Adell, A., Artigas, F., Boulton, A., Baker, G., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brain+microdialysis:+Principles+and+applications.">In vivo Brain microdialysis: Principles and applications.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Kendrick, K., Robinson, T., Justice, J., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+in+large+unrestrained+animals:+neuroendocrine+and+behavioral+studies+of+acetylcholine,+amino+acid,+monoamine+and+neuropeptide+release+in+the+sheep.">Microdialysis in large unrestrained animals: neuroendocrine and behavioral studies of acetylcholine, amino acid, monoamine and neuropeptide release in the sheep.</a> Microdialysis in the Neuroscience. 7, 327-348 (1991).</li><li>Oakley, B., Schafer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Neurobiology: A laboratory Manual. , (1978).</li><li>Okumura, T., Yamashita, Y., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+Finch+I.+A+Biological+study.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese Finch I. A Biological study.</a> Soc. Neurosci Abst. 33, (2007).</li><li>Boulton, A., Baker, G., Robinson, T., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relation+of+brain+electrical+activity+to+behavior.">The relation of brain electrical activity to behavior.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Yamashita, Y., Okumura, T., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+finch.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese finch.</a> , 101-104 (2007).</li></ol>

Experimentos foram realizados em conformidade com as orientações para a experimentação animal do cérebro Science Institute, RIKEN (RIKEN-BSI) e animal do instituto comitê de ética aprovado.

Em comparação com a aplicação de resinas acrílicas convencionais auto cura, a técnica introduzida tem várias vantagens importantes: 1. Ligação direta: Acrílica auto-cura pode ser conectado ao osso por um tempo mas não adere ao osso. Portanto, os parafusos de fixação são necessários para corrigir uma base de montagem (ver Figura 2a). Em contraste, a resina de cura pela luz pode vínculo até o osso diretamente de modo parafusos não são necessários (Figura 2B). Portanto, o tamanho da base de montagem pode ser reduzido consideravelmente. Além disso, as resinas de cura pela luz também adere a alguns metais, se você usou &quot;primer metal&quot; com ele. Esta característica da ligação a materiais variável permite uma ampla gama de aplicação. A Figura 2 mostra uma implantação usando acrílicos auto convencionais de cura (em 2A) e um implante com luz resinas adesivas de cura (no 2B). Em 2A, o parafuso de âncora é utilizada para fixar a base de montagem. Em 2B, o tamanho da base de montagem pode ser reduzida consideravelmente devido ao fato de que a resina de cura pela luz aderem ao crânio e parafusos como não são necessários. <img src="/files/249_2008_8_26_4/249_Figure-02.png" /> Figura 2 2. Fácil de manusear: Janela de resina convencional o tempo de processamento é relativamente curto, em poucos minutos. Ele tem uma alta fluidez e não é fácil de manusear à primeira vista. Entretanto, uma vez iniciados reação química, a reação é acelerada pelo calor exotérmica e resinas se tornam mais difíceis em pouco tempo. As janelas de tempo de processamento para resina de fotopolimerização são significativamente mais larga e ajustável. Basicamente, as resinas manter elasticidade constante desde o início até o momento em que é fotopolimerizado. 3. Reação não gera calor: Ea reação de cura da resina autopolimerizável é exotérmica. Quando a base de montagem é de espessura, este calor acumula e pode danificar o tecido. Reação da resina de cura pela luz não gera calor eo material é biocompatível. 4. Feitas à mão luz LED: Custo do material e equipamentos especiais, como unidade de luz pode ser um problema. Na Figura 3, apresentamos uma mão feita LED unidade de luz que não custa muito. Esta mão-made unidade de fotoativação podem ser usados ​​como substitutos para a unidade comercial caro. Figura 3A mostra um diagrama de circuito da unidade de luz. Figura 3B mostra três LEDs colados paralelos uns aos outros, e inserido no suporte de um marcador quadro branco. <img src="/files/249_2008_8_26_6/249_Figure-03.png" /> Figura 3 - Diagrama do circuito (em A) e fotografia (em B) de uma unidade hand-made de luz Esta técnica é aplicável a implantes de eletrodos crônica ou sonda de microdiálise em outros animais de experimentação, tais como peixes, mouse, coelho, gato e macaco.

1A fotografia mostra sonda de microdiálise (a), um tampão (b) e uma cânula guia (c). Nesta foto, a cânula guia é cercado por resina dental que adere ao crânio diretamente. Foto B mostra um pássaro durante a perfusão de drogas. No topo da cabeça, há uma sonda de microdiálise que é conectado a um tubo tefron bem. Barra de escala = 1 cm para A e 2 cm de B, respectivamente. <img src="https://www-jove-com.remotexs.ntu.edu.sg/files/249_2008_8_26_3/249_Figure-01.png" /> Figura 1 - Fotografias de uma sonda de microdiálise (A) e um pássaro durante a perfusão de drogas (B)

application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

Em experimentos de gravação crônica, auto-cura dental resinas acrílicas têm sido usados ​​como uma base de montagem de eletrodos ou sondas de microdiálise-. Uma vez que estes não acrílicos vínculo até o osso, parafusos têm sido usados ​​como âncoras. No entanto, em pequenos animais experimentais como tentilhões ou mouse, seus crânios são muito frágeis e não podem realizar com sucesso as âncoras. Neste relatório, propomos uma nova aplicação de luz de cura dental para as resinas base de montagem de eletrodos ou sondas de microdiálise em experimentos crônica. Este material permite a ligação directa ao crânio. Portanto, parafusos de âncora não são necessários e campo cirúrgico pode ser reduzido consideravelmente. Experiências passadas mostram que o efeito de ligação mantém mais de 2 meses. Janela de resina convencional de tempo, quando os materiais são maleáveis ​​e viável é de alguns minutos. No entanto, a janela do tempo de trabalho para estes adesivos dentais é significativamente mais larga e ajustável.

Watch this Scientific Journal Video about Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments at JoVE.com

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Neste relatório, propomos uma nova aplicação de luz de cura dental para as resinas base de montagem de eletrodos ou sondas de microdiálise em experimentos crônica. Este material permite a ligação directa ao crânio.

Aplicação de Light-resina adesivo dental para Eletrodos de montagem ou Sondas microdiálise em Experimentos crônica

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

application-light-cured-dental-adhesive-resin-for-mounting-electrodes

Nanyang Technological University

Research

JoVE Journal

Biology

15.3K visualizações.  RIKEN. Neste relatório, propomos uma nova aplicação de luz de cura dental para as resinas base de montagem de eletrodos ou sondas de microdiálise em experimentos crônica. Este material permite a ligação directa ao crânio.

Vídeo: Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Preparação de Bicamadas Artificial para Experimentos Eletrofisiologia

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Biologia

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabricação de eletrodos amperométrica

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robust FISH DNA 3D utilizando sondas Diretamente rotuladas

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Transplantes faciais em<em&gt; Xenopus laevis</em&gt; Os embriões

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicamadas de montagem para longo prazo Microscopia de Luz Folha de Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissecção e montagem de<em&gt; Drosophila</em&gt; pupais Discos Eye

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

A Assembleia e Aplicação de &#39;tesoura Anéis&#39;: A endotelial novo modelo de Orbital, unidirecional e periódica fluxo de fluido e tensão de cisalhamento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Aplicação de codificados geneticamente fluorescente óxido nítrico (NO •) Sondas, as geNOps, para imagens em tempo real de NO • Os sinais em células individuais

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Um método versátil para a montagem de Arabidopsis deixa para Intravital imagem de lapso de tempo

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...