Biz bu çalışmaları sırasında nazik destekler ve verimli tavsiyeleri RIKEN-BSI meslektaşlarımıza teşekkür ederiz. Profesör Shigemi Mori, Profesör G bor Juh sz, Dr benim samimi takdirlerimi ifade etmek istiyorum. İleri Araştırmalar Yüksek Lisans Üniversitesi, Japonya lisansüstü ders sırasında bana mikrodiyaliz yöntemin tavsiye Kiyoji Matsuyama, Katalin AK kesi ve rp d Dobolyi. Son olarak, Sayın Chris Sarnecky ve Dr. Akitsugu Takasu video kaydı destekler için teşekkür ederim.

1. Adım. Prob implantasyon hazırlanması <ol><li> Anestezi (video gösterilmez) Interperitoneal ketamin enjeksiyonu (12.5 mg / kg vücut ağırlığı) ve xylazine (25 mg / kg) karışımı cerrahi anestezi istikrarlı bir seviyede üretmek için her kuş anestezisi. </li><li> Stereotaksik bir çerçeveye Fix bir kafa Xylocaine (TM) jöle, kulak bar basınç yerel ağrılarını azaltmak için kullanın . Kuş baş, kulak bar ve 45 ° açıyla kafa tutan bir gaga sahibinin oluşan bir stereotaksik aparat sabittir . Implantasyonu için stereotaksik cerrahi sırasında, termal kontrol ile cerrahi ped kuş vücut ılık (sıcaklık = 34 ~ 36 ° C). </li><li> Cerrahi alan dezenfekte edin Ameliyat öncesinde isodine (TM) ile cerrahi alanında dezenfekte edin. </li></ol> 2. Adım. Stereotaksik konumlandırma <ol><li> Referans noktası hizalaması Lambda (Y-noktası) ve işareti Y-nokta boya dolu bir pipet ile kukla prob (cam mikropipet) hizalayın . Kalan son tracer çalışma Dextran-TMR video kullanıldı. </li><li> Cilt insizyonu Cilt çıkarın ve kafatası yüzeyinde ortaya çıkarmak. Sonra, pamuklu çubukla kafatası yüzeyi temiz. </li><li> Kukla kanül yerleştirilmesi (yatay yönde) Hedef site (AP ekseni ve Yanal koordinasyon) kukla kanül taşıyın </li><li> Küçük kraniotomi ve dura mater kesi. Kraniotomi için iyi bir forseps ve duramater kesiler için ince bir enjeksiyon iğnesi (30 G) kullanın. Sonra, kafatası yüzeyi temizleyin. </li><li> Kukla kanül Final konumlandırma (implantasyon) Mikrodiyaliz prob amaçlanan hedef bölge üzerinde 0.30 mm kılavuz kanül ucu bulun. Son olarak, temiz ve kuru bir yüzey sert yapıştırılması için gerekli olduğundan, tekrar kafatası yüzeyi temiz. </li></ol> Adım 3. Kraniyal yüzey ön hazırlık (Diş ürün &quot;doğrudan dolgu restorasyonu&quot; için benzer bir prosedür istihdam edilecektir.) <ol><li> Primer tedavi <ol><li> Bir çanak B Mix Primar sıvı. </li><li> Kafatasına küçük bir fırça ile yüzey karışımı uygulayın. </li><li> 30 saniye bekletin. </li><li> Hafif hava akımı ile kurulayın. (Yıkamayın.) </li></ol></li><li> Bonding ajanı uygulaması <ol><li> Bonding ajanı kafatasına küçük bir fırça ile yüzey üzerinde uygulayın. </li><li> Hafif bir hava akımı uygulayın. (Yıkamayın.) </li><li> Hafif-20 saniye boyunca tedavi. </li></ol></li></ol> Adım 4. Montaj kompozit <ol><li> Mount düz plugger reçine Düz plugger mevcut değilse, bir mikro-spatül yerine kullanılabilir. </li><li> Işık kür </li><li> Artımlı montaj Artımlı montaj reçine tam reaksiyon sağlamak için tavsiye edilir. Artımlı montaj için, her montaj önce bonding ajanı geçerlidir. </li><li> Son sertleşme ve ışıkla sertleşen kompozit kalıp. </li><li> Ameliyat sonu </li></ol> Cerrahi alanı temizleyin (Gentamisin merhem) antibiyotik uygulamak ve stereotaksik alev hayvan kaldırmak. Sonra, bir termo-kontrollü odası (sıcaklık = 33-35 ° C) anestezi sonrası kurtarma için hayvan tutun. Videoda görüldüğü gibi, herhangi bir anormal davranış, ertesi gün ameliyat sonrası herhangi bir hayvan gözlendi.

<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>

Deneyler hayvanın beyin deney bilim enstitüsü, RIKEN (RIKEN-BSI) ve enstitünün hayvan etik kurul onaylı yönergelere uygun olarak yapılmıştır.

Geleneksel kendi kendini iyileştirme akrilik reçine uygulama ile karşılaştırıldığında, piyasaya teknik birkaç önemli avantajı vardır: 1. Doğrudan bağlama: Bir süre için kendi kendini iyileştirme akrilik kemik bağlı olabilir ama bu kemiğe bağlı değildir. Bu nedenle, ankraj vidaları (Bkz. Şekil 2A) bir montaj üssü düzeltmek için gereklidir. Buna karşılık, ışıkla sertleşen reçine kemik doğrudan vida gerekli değildir (Şekil 2B) bağ. Bu nedenle, montaj tabanı boyutu önemli ölçüde azaltılabilir. Ile &quot;metal astar&quot; Buna ek olarak, ışıkla sertleşen reçineler de, bazı metallerin yapışır. Değişken malzemelerin yapıştırılmasında Bu özellik bize, geniş bir uygulama yelpazesi sağlar. Şekil 2, konvansiyonel kendi kendini iyileştirme akrilik (2A) ve ışıkla sertleşen bir yapıştırıcı reçineler (2B) kullanan bir implantasyon kullanarak bir implantasyon gösterir. 2A, çapa vidalı montaj tabanı düzeltmek için kullanılır. 2B, ışıkla sertleşen reçine kafatası ve cıvata uymasını gerçeği nedeniyle montaj tabanının boyutunu önemli ölçüde azaltılmış olabilir gerekli değildir. <img src="/files/249_2008_8_26_4/249_Figure-02.png" /> Şekil 2 2. Kullanımı kolay: Konvansiyonel reçine işleme zaman penceresi birkaç dakika içinde, nispeten kısa. Bu yüksek bir akışkanlığa sahip ve ilk bakışta ele almak kolay değil. Ancak, kimyasal reaksiyon bir kez başladı, tepkime ekzotermik ısı ile hızlandırılmış ve reçineleri kısa bir süre içinde daha zor hale gelir. Işıkla sertleşen reçine için işlem süresi pencereler, önemli ölçüde daha geniş ve ayarlanabilir. Temel olarak, ışıkla sertleşen reçineler başından itibaren anı sürekli elastikiyetini tutmak. 3. Reaksiyon ısı üretmek değildir: Kendi kendine sertleşen reçine tepki Kür Ekzoterm. Montaj tabanı kalın olduğunda, bu ısı birikir ve doku hasarı olabilir. Reaksiyon ışıkla sertleşen reçine ısı oluşturmaz ve malzemenin biyouyumlu. 4. El LED ışık: Işık ünitesi gibi malzeme ve özel ekipman maliyeti, bir sorun olabilir. Şekil 3&#39;te, çok maliyetli olmayan bir el LED ışık ünitesi tanıtmak. Bu el yapımı hafif kavurma ünitesi pahalı ticari birim için ikame olarak kullanılıyor olabilir. Şekil 3A, ışık ünitesi devre şeması gösterir. Şekil 3B, birbirine paralel yapıştırılmış ve beyaz tahta marker sahibi takılı üç LED gösterir. <img src="/files/249_2008_8_26_6/249_Figure-03.png" /> Şekil 3 - Devre diyagramı el yapımı bir ışık ünitesi ve fotoğraf (B) (A) Bu teknik, balık, fare, tavşan, kedi ve maymun gibi diğer deney hayvanlarında elektrotlar ya da mikrodiyaliz prob kronik implantasyonu uygulanabilir.

Fotoğraf 1A mikrodiyaliz prob (a), bir kap somunu (b) ve (c) bir kılavuz kanül gösterir. Bu fotoğrafta, kılavuz kanül doğrudan kafatasına bağlı diş reçine ile çevrilidir. Fotoğraf B ilaç perfüzyon sırasında bir kuş gösterir. Başın üst kısmında ince bir Tefron tüpüne bağlı bir mikrodiyaliz prob vardır. Ölçeği bar B A ve 2 cm = 1 cm, sırasıyla. <img src="https://www-jove-com.remotexs.ntu.edu.sg/files/249_2008_8_26_3/249_Figure-01.png" /> Şekil 1 - mikrodiyaliz prob (A) ve ilaç perfüzyon sırasında bir kuş fotoğrafları (B)

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

Kronik kayıt deneylerde, kendi kendine sertleşen diş akrilik reçineler elektrotlar veya mikrodiyaliz-probları bir montaj üssü olarak kullanılmıştır. Bu akrilik kemik bağı olmadığından, vida çapa olarak kullanılmıştır. Ancak, ispinozlar veya fare gibi küçük deney hayvanlarında, kafatasları çok kırılgan ve başarılı bir çapa tutamaz. Bu raporda, ışıkla sertleşen diş reçine elektrotlar veya kronik deneylerde mikrodiyaliz probları montaj tabanı için yeni bir uygulama öneriyorum. Bu malzeme, kafatası doğrudan yapıştırma sağlar. Bu nedenle, demir vida gerekmez ve cerrahi alanında önemli ölçüde azaltılabilir. Geçmiş deneyimler, yapıştırma etkisi en fazla 2 ay koruduğunu göstermektedir. Konvansiyonel reçine pencere malzemeleri, esnek ve uygulanabilir bir kaç dakika. Ancak, bu diş yapıştırıcılar için çalışma süresi pencere, önemli ölçüde daha geniş ve ayarlanabilir.

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

Bu raporda, ışıkla sertleşen diş reçine elektrotlar veya kronik deneylerde mikrodiyaliz probları montaj tabanı için yeni bir uygulama öneriyorum. Bu malzeme, kafatası doğrudan yapıştırma sağlar.

Kronik Deneyleri Montaj Elektrotlar veya mikrodiyaliz Problar Işık tedavi Diş Yapıştırıcı Reçine Uygulama

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 Görüntüleme.  RIKEN. Bu raporda, ışıkla sertleşen diş reçine elektrotlar veya kronik deneylerde mikrodiyaliz probları montaj tabanı için yeni bir uygulama öneriyorum. Bu malzeme, kafatası doğrudan yapıştırma sağlar.

Video: 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.

Elektrofizyoloji Deneyler Yapay Bilayers hazırlanması

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

Biyoloji

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.

Amperometrik Elektrotlar imalatı

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.

Doğrudan Etiketli Problar kullanarak Sağlam 3D DNA BALIK

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.

Yüz Nakli<em&gt; Xenopus laevis</em&gt; Embriyolar

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.

Çok katmanlı Zebra balığı uzun vadeli Işık Sayfalık Mikroskopi için Montaj

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.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

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.

Diseksiyon ve montajı<em&gt; Drosophila</em&gt; Pupa Göz Diskler

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.

Montaj ve &#39;Kayma Rings&#39; Uygulama: Orbital tek yönlü ve Periyodik Akışkan Akışının ve Kayma Stres için Yeni Bir Endotel Modeli

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.

Genetik Kodlanmış Floresan Nitrik Oksit Uygulama (• NO) Tek Hücrelerde YOK • Sinyallerin Gerçek zamanlı Görüntüleme için sondalar, geNOps,

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.

Arabidopsis montaj için çok yönlü bir yöntem Intravital zaman hata görüntüleme için yaprakları

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