私たちは、霊長類の仕事の継続的なサポートのために、フランクエルヴィン、ロバータパーマー、セントキッツ、西インド諸島に位置する行動科学財団研究所のスタッフに感謝しています。ヘルスリサーチ（AC）とナショナルエンジニアリングとカナダの研究評議会（MP）のカナダの協会からオペレーティング助成金 - この作品は、カナダの脆弱X研究財団（FXRFC）からSZの親睦とによってサポートされていました。

Immunohistochemistyは、様々な実験動物モデルの脳内におけるタンパク質発現を特徴づけるための最も広く使用されている技術の一つです。同様の脳の大きさを持つ齧歯類や他の一般的な実験モデルの脳で体系的免疫組織化学的手続を実施するのは比較的簡単です。しかし、全体のサルの脳でそのような免疫検出の手順を実行する方法の包括的な報告を提供する我々の知識への公開作品はありません。以下は、様々な標的タンパク質の大規模な免疫組織化学的検出のために全体のサルの脳を準備する方法の詳細な説明です。この作品は、商用および学術的努力とのコラボレーションの結果として浮上している。このように、埋め込み、切片組織に関する詳細は、NSAの独自の知識のまま。 パート1：動物の治療や組織の準備成人のベルベットモンキー（ ミドリザル ）から脳を、本プロトコルに使用されます。すべての手順は、生物医学研究1の動物の利用とケアのためのアニマルケアに関するカナダの協議会（CCAC）ガイドラインに準拠して行われている。 <ol><li>動物は深く、（10 mg / kgを、IM）塩酸ケタミンで鎮静ペントバルビタールナトリウムの過剰投与（25 mg / kgを、IV）を使用して、安楽死させ、完全に放血するまで0.1MのPBSでtranscardially灌流される。 </li><li>これは、5分（〜1リットル）のためのPBS中4％パラホルムアルデヒド溶液が続いている。 </li><li>脳は、容器の底に沈む˚Cまで、外部化された脳は、0.02％アジ化ナトリウムを含む段階的PBS -バッファショ糖溶液（シーケンス10、20および30％）に入れ、4に維持される。脳は、凍結切片を経るまでのソリューションは、数日おきに置換されます。 </li><li>彼らの独自のMultiBrain™埋め込みおよび凍結セクショニング技術に供される、脳が神経科学アソシエイツ（ノックスヴィル、テネシー州NSA）に送信されます。 </li><li>セクションが適切な方向になる（すなわち、左から右方向）と組織学的処理後に再整列できるように、7（7）アライメントマークを埋め込むマトリックスに配置されます完了です。 </li><li>各シリアルセクションがブロックから収集される前に凍結ブロックは、デジタルブロック面で撮影されています。デジタル画像だけでなく、この脳からのセクションは、それぞれ後、解剖学的および組織学的マップの3D再構成のために使用することができます。 </li><li>シリアルフリーフローティング冠状セクション、セクションごとに50μmの厚さで、脳全体から収集されます。 </li></ol>第2部：組織学的処理セクションは、与えられた空間の間隔で選ばれている（例えば、500μm）と我々の実験目的のために以下の抗体で処理した：脆弱X精神遅滞タンパク質（FMRP、ケミコン、テメキュラ、カリフォルニア州）、SMI32（シュテルンベルガーモノクローナル抗体（株）;ボルチモア、MD）、およびNeuN（ケミコン、テメキュラ、カリフォルニア州）。 FMRPは豊富に正常と順列キャリアの脳の神経細胞2に見られる細胞質タンパク質である。 NeuN（神経核）は、特にほとんどのニューロンに存在し、神経核、perikaryonの複数形といくつかの近位の神経のプロセス3で配布されているDNA結合ニューロン特異的タンパク質のNeuNを認識します。 SMI - 32は、ニューロフィラメント蛋白質4の非リン酸化エピトープを認識するモノクローナル抗体である。 <ol><li> 500μmの間隔で、約140のセクションは、抗体ごとに使用されています。 </li><li>すべてのインキュベーション、洗浄および染色ステップは、特殊な染色皿やバスケット（;ノックスヴィル、テネシー州HistoTools）を用いて軽く攪拌しながら実施されています。 </li><li>セクションは、最初の抗体分子の非特異的結合と、その結果、バックグラウンド染色を低減するために、0.1 M PBS/0.3％トリトンX - 100（TX）/ 60分間5％正常しわがれ血清（NHS）を含む溶液中でインキュベートする。 </li><li>ベンダーの指示に従って、FMRPの免疫染色用にマークされたセクションでは、抗原のアンマスキングソリューション（バーリンゲーム、カリフォルニア州ベクトルラボ）を使用して、抗原回復の追加のステップを経る。 </li><li>セクションは、NeuN抗体について（1 / 5の希釈率で、FMRPとSMI - 32と1 / 2、000の000、0.1 M PBS/0.3％TX / 3％NHSを含む、それぞれの抗体溶液中で一晩とは別の日にインキュベートされる。 </li><li> 1 /;次の日は、セクションは馬で提起されたビオチン化抗マウス二次抗体で室温で2時間インキュベートした洗浄液で3つの10分の期間（0.1 M PBS/0.3％TX）（ベクター研究所のために洗浄されています0.1 Mの1,000倍希釈PBS/0.3％TX / 3％NHS）。 </li><li> 3 10分間の洗浄のさらなるセットの後、切片を室温で1時間アビジン - ビオチン結合した西洋ワサビペルオキシダーゼ複合体（ベクター研究所）の溶液中に配置されます。 </li><li>最後に、洗浄の別のセットの後、切片をニッケル強化された径に10分間さらされているminobenzidine（ニッケルDAB）免疫反応性ニューロン内の染色ダークブルーを生成する反応。 </li><li>組織学的手順の完了時に、セクションは、ゼラチンコートしたスライドガラス上にマウントされています。 </li><li>すべてのセクションでは、一晩風乾しており、標準的な手順に従って封入。 </li></ol>パート3：代表的な結果： このメソッドは、全体のサルの脳全体の関心の標的タンパク質の完全な発現プロファイルを生成します。ここではFMRP、NeuNと同じサルの脳内SMI32式のスナップショットを提供する代表的な冠状セクションを示しています。 <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> 図1：免疫検出のためのフリーフローティングセクションの大規模なバッチ処理に使用される染色皿（）とバスケット（B）。 <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> 図2：FMRP（）、NeuN（B）とSMI32（C）抗体で染色ベルベットモンキーの脳からの代表的な冠状断面。

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

可能な限り全体のサルの脳内のタンパク質の大規模な検出を行うこの手順の2つの重要なステップがあります。一つは、NSAの独自の知識のまま埋め込むとセクショニングのプロトコルです。他はHistoToolsで提供される染色料理やバスケットを使用することです。後者は、所定の時間に多くの（〜40）のセクションの容易かつ迅速な処理を行うことができます。また、一様に脳全体のすべてのセク...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>anti-FMRP monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB2160</td>
<td>Requires antigen retrieval in fixed tissue.</td>
</tr>
<tr>
<td>anti-NeuN monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB377</td>
<td>N/A</td>
</tr>
<tr>
<td>anti-Neurofilament H monoclonal antibody</td>
<td>&nbsp;</td>
<td>Sternberger Monoclonals Inc.</td>
<td>SMI32</td>
<td>N/A</td>
</tr>
<tr>
<td>Antigen Unmasking Solution</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-3300</td>
<td>N/A</td>
</tr>
<tr>
<td>Normal horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050122</td>
<td>500 mL</td>
</tr>
<tr>
<td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>BA-2000</td>
<td>&nbsp;</td>
<tr>
<td>VECTASTAIN ABC Kit (Standard)</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>PK-4000</td>
<td>1.5 mg</td>
</tr>
<tr>
<td>Staining dish for floating sections</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10009</td>
<td>12 x 65 mm</td>
</tr>
<tr>
<td>Staining transport basket</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10012</td>
<td>large 
(fits 12 x 65 mm dish)</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>P8514B</td>
<td>N/A</td>
</tr>
<tr>
<td>DAB</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC11209-0050</td>
<td>N/A</td>
</tr>		</tbody>
		</table>
		</div>

batch immunostaining for large-scale protein detection in the whole monkey brain

免疫組織化学（IHC）は、標的タンパク質の検出のための最も広く使用されている実験手法の一つです。<em&gt;その場で</em&gt;。脳全体にわたる標的タンパク質の発現パターンに関する質問は、このようなげっ歯類のものなどの小さな脳でIHCを使用するときに答えることが比較的容易である。しかし、そのような霊長類のものとして大規模かつ複雑な脳の同じ質問に、答えることは多くの課題を提示。ここでは、成人のサルの脳内の標的タンパク質の免疫検出のための体系的なアプローチを提示する。このアプローチは、神経科学アソシエイツ（NSA）と同様に自由に浮遊切片のバッチ染色のために特別に開発されたツールの方法論を埋め込み、セクションなどの組織に依存しています。それは特定の間隔で、脳全体を表す、セクションの集合の一様な染色をもたらす。結果として染色した切片は、特定のタンパク質を発現するニューロンの人口、タンパク質レベルを測定するために分析的手続の多種多様に供することができる。

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

全体の霊長類の脳で標的タンパク質の大規模な免疫検出は、任意の時点で複数のフリーフローティングセクションのバッチ染色のための創造的な装置の使用を組み合わせる方法を埋め込み、セクションなどの新たな組織を採用することで可能です。

全モンキー脳の大規模なタンパク質の検出のためのバッチの免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

batch-immunostaining-for-large-scale-protein-detection-whole-monkey

Research

JoVE Journal

Biology

12.6K ビュー.  Montreal Neurological Institute. 全体の霊長類の脳で標的タンパク質の大規模な免疫検出は、任意の時点で複数のフリーフローティングセクションのバッチ染色のための創造的な装置の使用を組み合わせる方法を埋め込み、セクションなどの新たな組織を採用することで可能です。

ビデオ: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

大規模ゼブラフィッシュベース in vivoで小分子スクリーン

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

大規模なSNPジェノタイピングアプリケーション用インフィニウムアッセイ

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感な、大規模定量的メタボロームのための戦略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY：酵母増殖の多パラメータ定量化のための大規模法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

大精子細胞の3D共焦点解析のための全山 ショウジョウバエ 精巣の免疫染色

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...