資金は、NSF CCLI番号0633618とオハイオ大学によって提供されました。いくつかの株は、線虫の遺伝学センターから提供された。大学院生Reetobrata Basuさんは、ビデオに表示されます。

<ol>
	<li>Harlow, E., Lane, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibodies: A Laboratory Manual. , (1988).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Transformation.">DNA Transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Boulin, T., Etchberger, J. F., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene+fusions.">Reporter gene fusions.</a> WormBook. , (2006).</li><li>Duerr, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> WormBook. , (2006).</li><li>Finney, M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unc-86+gene+product+couples+cell+lineage+and+cell+identity+in+C.+elegans.">The unc-86 gene product couples cell lineage and cell identity in C. elegans.</a> Cell. 63, 895-905 (1990).</li><li>Garriga, G., Desai, C., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+control+the+direction+of+outgrowth,+branching+and+fasciculation+of+the+HSN+axons+of+Caenorhabditis+elegans.">Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans.</a> Development. 117, 1071-1087 (1993).</li><li>Miller, D. M., Shakes, D. C., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Caenorhabditis elegans: Modern Biological Analysis of an Organism. , 365-394 (1995).</li><li>Crittenden, S. L., Troemel, E. R., Evans, T. C., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLP-1+is+localized+to+the+mitotic+region+of+the+C.+elegans+germ+line.">GLP-1 is localized to the mitotic region of the C. elegans germ line.</a> Development. 120, 2901-2911 (1994).</li><li>Strome, S., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+visualization+of+germ-line-specific+cytoplasmic+granules+in+embryos,+larvae,+and+adults+of+Caenorhabditis+elegans.">Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 79, 1558-1562 (1982).</li><li>Albertson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+first+cleavage+spindle+in+nematode+embryos.">Formation of the first cleavage spindle in nematode embryos.</a> Dev. Biol. 101, 61-72 (1984).</li><li>Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using Antibodies: A Laboratory Manual. , (1999).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> WormBook. , (2006).</li></ol>

著者は、彼女が競合する経済的利益を持っていないことを宣言します。

凍結割れプロトコルは、Cの抗体染色のためのいくつかの方法の一つである虫。これは、ワームを染色するための比較的簡単な方法を提供していますが、最適な結果を得るための具体的な試薬と実践を必要としません。 （ 図1に概要）の重要なステップは、1）ステップ1と線虫の2）マニュアルの圧縮（ステップ2および3を参照のこと）迅速な固定をする（ステップ3）?...

タンパク質の細胞と細胞内局在を決定するために、科学者たちは、伝統的には特定のタンパク質1を認識するように選択された抗体を有する組織のラベルが付いています。 Cなどのいくつかのモデル生物でのエレガンス 、抗体染色は、多くの場合、より迅速に結果が得られた分子遺伝学的技術、により置換されている。これらは、プロモーターと関心と緑色蛍光タンパク質の遺伝子のコード領域の間の遺伝子の融合からなる構築物で形質転換する生物を含む。しかし、分子技術は、真のプロモーターと高コピー数（C. elegansの通常の技術） の2,3での構築物の発現の変化を知ることで問題を含むアーティファクトの数、対象となります。したがって、抗体を用いた染色は、生体内でのタンパク質の機能を研究する多くの科学者のための目標である。 抗体を用いて組織を染色することは、困難な場合がありますので、tibodiesは、特定の立体構造1にそれらの抗原を認識することができる。例えば、抗体は、特定の方法で修正されただけの変性または完全な抗原を認識することができるし、 その場で抗原を認識しない場合があります。線虫における抗体染色の問題は、線虫のクチクラは、組織への抗体のアクセスをブロックし、比較的不透過性のバリアを形成するという事実によって悪化する。 Cの抗体染色のために使用される、いくつかの異なる方法があります。 （私たちの以前の研究4に概説されている） 虫 。無傷で染色する「ワームを、「法は凍結し、比較的硬い固定液（ホルムアルデヒド、グルタルアルデヒド）で解凍するために開発された、凍結融解サイクルは、固定液5の急速な浸透を可能にするためにキューティクルをクラックするのに役立ちます。固定後、キューティクルは、抗体の浸透を可能にするために透過処理した;方法は、薬剤、コラゲナーゼ、またはその両方を低減5-7で処理することを含んでいた。これらのTreatmentsは、形態を保ったが、多くの場合、抗体認識を減少または破壊された。代替方法は、抗体の浸透を可能にするために解剖8が含まれています。 「凍結割れを「抗体染色9月10日を可能にするために半無傷のワームの内部へのアクセスを得るための一つの方法です。コラゲナーゼおよび還元処置を避けながら、凍結割れが固定の様々な条件で行うことができる。線虫は、2接着剤スライドの間に配置された凍結した後、スライドを分離し、下部のスライド上の線虫のほとんどは、トップスライド上のキューティクルの多くを残している。下部スライドは固定剤に配置することができ、付着した線虫を有するスライド全体を、抗体染色手順を介して転送される。この方法は、2つの主要な困難を提示しません。まず、真剣にそれらを変形させることなく、線虫を分割するのに必要な圧力の正確な量を適用することは困難である。第二に、ワームの多くはSではないでしょういずれかのスライドにチェックを入れて、固定液やリンスに失われます。しかし、適切なスライドと実践では、この方法は急速に固定剤および抗体は、多種多様で使用することができる合理的な形態を有する線虫を生じる。 この方法は、実験の目的に応じてわずかに変化させることができる。単一線虫の染色（ 例えば 、単一の形質転換体は、抗体染色を変化させたか否かを決定するために）所望される場合、最大の密着性と摺動する（しかし、より高いバックグラウンド染色）は、（下記参照）、このような余分なポリリジンを有する実験室で調製されたスライドを使用することができる。線虫がこれら注視後にポリリジンにもっと悪い付着するので、ホルムアルデヒドまたはグルタルアルデヒド固定のため、高い密着性スライド（実験室用意ポリリジンスライド）が、使用されるべきである。野生型線虫は、メタノールおよび/またはアセトンで固定されている場合は、下側の接着性と摺動し、低いバックグラウンド染色を用いるべきである（市販またはlaboratoryに準備スライド）。抗体および固定剤の種々の研究室で使用されている場合、必要​​になるまで、スライドの選択は、前もって調製し、保存することができる。 線虫の組織へのアクセスが獲得された後、抗体染色手順は、（より長いインキュベーション組織の特性よりもむしろ細胞1,11で）標準的な方法に従う。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine slide (ESCO brand)</td>
			<td>Fisher</td>
			<td>12-545-78</td>
			<td>Sigma brand slides also suitable</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P1524</td>
			<td>IMPORTANT: Brand and high molecular weight of polylysine critical</td>
		 </tr>
		 <tr>
			<td>single frosted edge slides</td>
			<td>Fisher</td>
			<td>48312-002</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S2002-25G</td>
			<td>TOXIC, wear gloves and mask while weighing out powder to make 10% stock. Wear gloves when pipeting stock solution</td>
		 </tr>
		 <tr>
			<td>coplin staining jar</td>
			<td>VWR</td>
			<td>47751-792</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>5-slide mailer</td>
			<td>Electron Microscopy Science</td>
			<td>71549-01</td>
			<td>One of many different brands of small slide holder, suitable for staining</td>
		 </tr>
		 <tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>MK262159</td>
			<td>IMPORTANT: Regular formalin solutions (37% formaldehyde) are NOT suitable. Either 1) Dissolve crysatlaline paraformaldehyde in phosphate buffer and store in small aliquots frozen. Or 2) buy electron microscopy grade paraformaldehyde in aliquots. TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Glutaraldehyde solution, 25% in water</td>
			<td>Sigma</td>
			<td>G 5882</td>
			<td>IMPORTANT: purchase electron microscopy grade glutaraldehyde in ampules; TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>EM-9410</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>ICN820451</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Donkey Serum, lyophilized</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Cy3 Donkey anti-Mouse IgG multi-labeling</td>
			<td>Jackson Immunoresearch</td>
			<td>715-165-150</td>
			<td>This company is recommended for high quality multi-label (affinity depleted) antibodies. Select appropriate dye and antigen (animal used to raise primary antibody). </td>
		 </tr>
		 <tr>
			<td>n-propyl gallate</td>
			<td>Fisher</td>
			<td>ICN10274750</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride</td>
			<td>Sigma</td>
			<td>D 9542</td>
			<td>TOXIC. Wear gloves and dispense into small aliquots and feeze to minimize exposure</td>
		 </tr>
		 <tr>
			<td>Whatman #1 15 cm diameter filters</td>
			<td>Fisher</td>
			<td>09805G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip #1 1/2 rectangle 24 x 60 mm</td>
			<td>VWR</td>
			<td>48393-252</td>
			<td>#1 1/2 thickness is optically best</td>
		 </tr>
		 <tr>
			<td>Microscope slide folder</td>
			<td>VWR</td>
			<td>48429-092</td>
			<td>Other brands are suitable</td>
		 </tr>
		</tbody>
 </table>

antibody staining in c. elegans using &quot;freeze-cracking&quot;

C.を染色する抗体を用いた虫は 、比較的不透過性のキューティクルは、化学的または機械的な方法でバイパスする必要があります。 「凍結割れを「物理的に二つのスライド間の接着性の線虫を圧縮し、それらを凍結し、そして離れてスライドを引っ張ることにより線虫のクチクラをプルするために使用される一つの方法である。凍結割れは、化学処理をせずに、組織へのアクセスを獲得するための簡単​​かつ迅速な方法を提供し、様々な固定剤と共に使用することができる。しかしながら、標本の多くの損失をもたらし、必要な圧縮は、機械的にサンプルを歪める。実際には、良好な形態を有するサンプルの回収を最大化するために必要とされる。凍結割れは、特定の固定条件、​​サンプルの回収、または低非特異的染色のために最適ではなく、すべてのパラメータの一度にすることができます。非常に難しいの固定条件を必要とし、化学的にキューティクルを透過性にするために必要な化学処理を容認する抗体について、treatmen溶液中のインタクトな線虫のtが好ましい場合がある。抗体が必要な場合は軽量化の修正や最適な固定条件が不明である場合には、凍結割れが急速に抗体をアッセイすると、目的の抗原に特異的な細胞内および細胞内局在情報を得ることができる非常に便利な方法を提供します。

ワームが適切に圧縮ひび割れ、軽く固定した場合、事実上、全体のワームは、抗体染色がアクセスすることができます（ 図2を参照）。 DAPI染色によって示されるように、核の位置は、ワームの部分がワームをホルムアルデヒドなどの硬い固定液に供された場合の不自然波状の出現によって示されるように、ワームの形態は（歪む無傷であるかを示す図3A〜Dの筋肉）?...

Watch this Scientific Journal Video about Antibody Staining in C. Elegans Using "Freeze-Cracking" at JoVE.com

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

「凍結割れ、「線虫C.の内部組織を露出させるための方法をタンパク質の局在化のための抗体に虫が 、実証されている。

抗体染色中<em&gt; C.虫</em&gt;「フリーズクラッキング」使用

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

antibody-staining-in-c-elegans-using-freeze-cracking

Research

JoVE Journal

Biology

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

22.4K ビュー.  Ohio University.  「凍結割れ、「線虫C.の内部組織を露出させるための方法をタンパク質の局在化のための抗体に虫が 、実証されている。 

ビデオ: Antibody Staining in C. Elegans Using "Freeze-Cracking"

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

マイクロインジェクションを使用して安定したトランスジェニック線虫の世代

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

イメージング C.エレガンス</em落射蛍光顕微鏡とオープンソースソフトウェアを使用して>胚

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

で遺伝子発現プロファイルを決定 C.エレガンスマイクロアレイとリアルタイムPCRを用いて

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegansの走化性アッセイ

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

における虚血/再灌流のための無酸素飢餓モデル<em&gt; C.虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

創傷応答のためのスキン傷つけ処理のための方法およびアッセイ<em&gt; C。エレガンス</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

の発達過程を追跡し、定量化<em&gt; C。エレガンス</em&gt;オープンソースのツールを使用しました

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

エレガンスにおける生理的ストレス応答の測定

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...