Wir danken unseren Kollegen in RIKEN-BSI für die freundliche Unterstützung und fruchtbare Ratschläge bei dieser Arbeit. Ich möchte meinen aufrichtigen Dank Professor Shigemi Mori, Professor G bor Juh sz, Drs. auszudrücken. Kiyoji Matsuyama, Katalin AK kesi und rp d Dobolyi, die mich zu Mikrodialyse-Methode während der Diplom-Studium an der Graduate University for Advanced Studies, Japan-Studie beraten. Abschließend danken wir Herrn Chris Sarnecky und Dr. Akitsugu Takasu für ihre Unterstützung in Video-Aufzeichnung.

Schritt 1. Vorbereitung der Sonde Implantation <ol><li> Anästhesie (nicht im Video gezeigt) Anesthetize jeder Vogel mit einem interperitoneale Injektion eines Ketamin (12,5 mg / kg Körpergewicht) und Xylazin (25 mg / kg) Mischung auf einem konstanten Niveau von chirurgischen Anästhesie zu erzeugen. </li><li> Fix einen Kopf zu einem stereotaktischen Rahmen Verwenden Sie Xylocain (TM) Gelee zu lokalen Schmerzen Ohr Bars &quot;Druck zu reduzieren. Der Kopf des Vogels ist in einem stereotaktischen Apparatur, bestehend aus Ohr Bars und einen Schnabel Halter, dass der Kopf in einem Winkel von 45 ° hält fixiert. Während der stereotaktischen Operation zur Implantation, erwärmen den Körper des Vogels durch chirurgische Pad mit thermischen (Temperatur = 34 ~ 36 ° C). </li><li> Desinfizieren OP-Bereich Desinfizieren Sie das OP-Feld mit isodine (TM) vor der Operation. </li></ol> Schritt 2. Stereotaktische Positionierung <ol><li> Referenzpunkt Ausrichtung Richten Sie Dummy-Sonde (Glasmikropipette), um die Lambda (Y-Punkt) und Mark Y-Punkt mit dem Farbstoff gefüllten Pipette. Verbleibende Dextran-TMR der letzten Tracer-Studie wurde in dem Video verwendet. </li><li> Inzision der Haut Entfernen Sie Haut und setzen der Oberfläche des Schädels. Dann reinigen Sie die Oberfläche des Schädels mit einem Wattestäbchen. </li><li> Die Positionierung der Dummy-Kanüle (in horizontaler Richtung) Bewegen Sie den Dummy-Kanüle an den Zielort (AP-Achse und Lateral-Koordination) </li><li> Kleine Kraniotomie und Inzision der Dura mater. Verwenden einer feinen Pinzette für Kraniotomie und einer Geldstrafe Injektionsnadel (30 G) für Einschnitte der Dura mater. Dann reinigen Sie die Oberfläche des Schädels. </li><li> Endgültige Positionierung (Implantation) von Dummy-Kanüle Suchen Sie die Spitze des Führungskanüle auf 0,30 mm über dem Mikrodialysesonde-anvisierten Region. Schließlich ist die Oberfläche des Schädels wieder, denn eine saubere und trockene Oberfläche für den harten Bindung erforderlich ist. </li></ol> Schritt 3. Eine Vorbehandlung der Oberfläche der Schädelbasis (Die ähnliche Vorgehensweise für &quot;direkte Befüllung Wiederherstellung&quot; der zahnärztlichen Produkt wird eingesetzt werden.) <ol><li> Primer-Behandlung <ol><li> Mix PRIMAR Flüssigkeit A mit B auf einem Teller. </li><li> Übernehmen Sie die Mischung auf der Oberfläche des Schädels mit einem kleinen Pinsel. </li><li> Lassen Sie für 30 Sekunden. </li><li> Trocken mit milder Luftstrom. (Nicht waschen.) </li></ol></li><li> Die Anwendung des Bindemittels <ol><li> Wenden Sie das Bindemittel an der Oberfläche des Schädels mit einem kleinen Pinsel. </li><li> Tragen Sie einen sanften Luftstrom. (Nicht waschen.) </li><li> Light-Kur für 20 Sekunden. </li></ol></li></ol> Schritt 4. Montage Komposit <ol><li> Berg-Harz mit einfachen plugger Wenn ein Plain Stopfer nicht verfügbar ist, kann ein Mikro-Spatel verwendet werden. </li><li> Lichthärtung </li><li> Incremental Montage Incremental Montage zu gewährleisten, eine vollständige Umsetzung des Harzes empfohlen. Bei inkrementellen Montage gelten Haftvermittler vor jeder Montage. </li><li> Mold den Komposit-und lichthärtenden für die endgültige Aushärtung. </li><li> Ende der Operation </li></ol> Reinigen Sie den OP-Bereich, gelten Antibiotikum (Gentamicin Salbe), und entfernen Sie das Tier aus der stereotaktischen Flamme. Dann halten die Tiere in einem thermo-kontrollierten Kammer (Temperatur = 33-35 ° C) für post-Aufwachraum. Wie in dem Video zu sehen, wurde kein anormales Verhalten in einem der Tiere am nächsten Tag nach der Operation beobachtet.

<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>

Die Experimente wurden in Übereinstimmung mit den Richtlinien für Tierversuche der Brain Science Institute, RIKEN (RIKEN-BSI) und des Instituts Tier Ethikkommission genehmigt ihnen unternommen.

Im Vergleich zu der Anwendung der konventionellen Selbsthilfeeffekt Acrylharze, hat die Einführung Technik mehrere wichtige Vorteile: 1. Eine direkte Verbindung: Selbst Akrylat kann bis auf die Knochen für eine Weile befestigt werden aber nicht bis auf die Knochen halten. Daher sind Verankerungsschrauben erforderlich, um eine Grundplatte fixieren (siehe Abbildung 2a). Im Gegensatz dazu kann Lichthärtung Kunstharzbindung bis auf die Knochen direkt so Schrauben sind nicht erforderlich (Abbildung 2B). Deshalb können Größe der Grundplatte erheblich reduziert werden. Darüber hinaus lichthärtenden Kunststoff hält auch einige Metalle, wenn Sie &quot;Metall-Primer&quot; verwendet mit ihm. Diese Funktion der Bindung an variable Materialien ermöglicht es uns, eine breite Palette von Anwendungen. Abbildung 2 zeigt eine Implantation mit herkömmlichen Selbsthilfeeffekt Acryl (in 2A) und eine Implantation mit Hilfe von Licht härtende Klebstoff-Harzen (in 2B). In 2A wird der Anker Schraube verwendet werden, um die Montageplatte befestigen. In 2B, Größe der Grundplatte kann erheblich durch die Tatsache, dass lichthärtenden Harz zum Hinterhaupt und solche Schrauben halten reduziert werden sind nicht erforderlich. <img src="/files/249_2008_8_26_4/249_Figure-02.png" /> Abbildung 2 2. Leicht zu handhaben: Konventionelle Harzes Verarbeitung Zeitfenster ist relativ kurz, innerhalb von wenigen Minuten. Es hat eine hohe Fließfähigkeit und es ist nicht leicht, auf den ersten Griff. Sobald jedoch chemische Reaktion gestartet, ist die Reaktion von exothermen Wärme beschleunigt und Harze schwieriger geworden, in kurzer Zeit. Die Verarbeitung Zeitfenster für lichthärtende Harz sind deutlich breiter und einstellbar. Grundsätzlich halten Harze konstante Elastizität von Anfang bis zu dem Moment, wenn es Licht ausgehärtet wird. 3. Reaktion keine Wärme: Härtungsreaktion der Autopolymerisat ist exotherm. Wenn die Grundplatte dick ist, sammelt sich diese Wärme und können das Gewebe beschädigen. Reaktion von lichthärtenden Harz erzeugt keine Wärme und das Material ist biokompatibel. 4. Handgemachte LED-Licht: Kosten des Materials und spezielle Ausrüstung wie Licht-Einheit kann ein Problem sein. In Abbildung 3 stellen wir eine handgefertigte LED-Einheit, die kostet nicht viel. Diese Hand-made Lichthärtegerät kann als Ersatz für teure kommerzielle eingesetzt werden. 3A zeigt ein Schaltbild Leuchteinheit. Abbildung 3B zeigt drei LEDs parallel miteinander verklebt, und in den Halter eines weißen Board-Marker. <img src="/files/249_2008_8_26_6/249_Figure-03.png" /> Abbildung 3 - Schaltbild (in A) und fotografieren (in B) eine Hand-made Leuchteinheit Diese Technik ist für chronische Implantation von Elektroden oder Mikrodialysesonde in anderen Versuchstieren wie Fisch, Maus, Kaninchen, Katze und Affe.

Foto 1A zeigt Mikrodialysesonde (a), einer Überwurfmutter (b) und eine Anleitung Kanüle (c). Auf diesem Foto ist die Führung Kanüle durch zahnärztliche Harz, das an den Schädel hält direkt umgeben. Foto B zeigt ein Vogel während der Droge Perfusion. Auf der Oberseite des Kopfes, gibt es eine Mikrodialysesonde, dass zu einer Geldstrafe tefron Rohr verbunden ist. Maßstabsbalken = 1 cm für A-und 2 cm für B sind. <img src="https://www-jove-com.remotexs.ntu.edu.sg/files/249_2008_8_26_3/249_Figure-01.png" /> Abbildung 1 - Fotografien einer Mikrodialyse-Sonde (A) und ein Vogel während der Droge Perfusion (B)

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

Bei der chronischen Aufnahme-Experimente haben selbsthärtenden zahnärztliche Acrylharzen als Grundplatte von Elektroden oder Mikrodialyse-Sonden verwendet worden. Da diese Acrylfarben nicht Bindung an den Knochen haben Schrauben als Anker benutzt worden. Allerdings in kleinen Versuchstieren wie Finken oder Maus, sind ihre Schädel sehr zerbrechlich und kann nicht erfolgreich halten die Anker. In diesem Bericht schlagen wir eine neue Anwendung von lichthärtenden Dentalharze für Montagesockel von Elektroden oder Mikrodialysesonden in chronischen Experimenten. Dieses Material ermöglicht die direkte Anbindung an die Schädeldecke. Daher sind Ankerschrauben nicht erforderlich und chirurgischen Bereich erheblich reduziert werden. Die bisherigen Erfahrungen zeigen, dass die Klebewirkung mehr als 2 Monate hält. Konventionelle Harzes Zeitfenster, wenn die Materialien weich und bearbeitbar sind nur ein paar Minuten. Allerdings ist das Fenster der Arbeitszeit für diese Dentaladhäsive deutlich breiter und einstellbar.

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

In diesem Bericht schlagen wir eine neue Anwendung von lichthärtenden Dentalharze für Montagesockel von Elektroden oder Mikrodialysesonden in chronischen Experimenten. Dieses Material ermöglicht die direkte Anbindung an die Schädeldecke.

Anwendung von Licht-gehärteten Dental Adhesive Resin für Montage-Elektroden oder Mikrodialysesonden in Chronic Experimente

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 Aufrufe.  RIKEN. In diesem Bericht schlagen wir eine neue Anwendung von lichthärtenden Dentalharze für Montagesockel von Elektroden oder Mikrodialysesonden in chronischen Experimenten. Dieses Material ermöglicht die direkte Anbindung an die Schädeldecke.

Serie: 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.

Herstellung künstlicher Doppelschichten für Elektrophysiologie Experimente

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

Forschung

Biologie

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.

Herstellung von amperometrische Elektroden

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 3D DNA FISH Mit Direkt markierten Sonden

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.

Gesichtstransplantation in<em&gt; Xenopus laevis</em&gt; Embryonen

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.

Multilayer-Halterung für Langzeit-Licht-Mikroskopie Blatt der Zebrafisch

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.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

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.

Dissection und Montage<em&gt; Drosophila</em&gt; 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 ...

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.

Die Montage und Anwendung der &quot;Shear Ringe &#39;: A Novel Endothelial Modell für Orbital, unidirektional und periodische Strömungs- und Scherspannung

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.

Die Anwendung von genetisch kodierter Fluorescent Stickstoffmonoxid (NO) • Sonden, die geNOps, für Echtzeit-Imaging von NO • Signale in Einzelzellen

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.

Eine vielseitige Methode für die Montage von Arabidopsis verlässt für intravitalen Zeitraffer Aufnahmen

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