Nous sommes reconnaissants à Frank Ervin, Roberta Palmour et le personnel des laboratoires de sciences du comportement de la Fondation situé à St-Kitts, dans les Antilles, pour leur soutien continu de notre travail primates. Ce travail a été soutenu par une bourse de la Fondation X Fragile de recherches du Canada (FCRSXF) pour la SZ et - les subventions de fonctionnement des Instituts canadiens de recherche en santé (AC) et le National de l&#39;Ingénierie et de Conseil de recherches du Canada (MP).

Immunohistochimie est l&#39;une des techniques les plus utilisées pour la caractérisation de l&#39;expression des protéines dans le cerveau de différents modèles animaux expérimentaux. Il est relativement facile à mener systématiquement des procédures immunohistochimiques sur le cerveau des rongeurs et autres communes des modèles expérimentaux avec la taille du cerveau similaires. Cependant, il n&#39;ya pas de travaux publiés, à notre connaissance qui fournit un compte rendu exhaustif de la façon de mener les procédures immunodétection tels travers un cerveau de singe entier. Ce qui suit est une description détaillée de la façon de préparer un cerveau de singe toute à grande échelle détection immunohistochimique des protéines cibles. Ce travail est apparu comme un résultat de la collaboration entre les activités commerciales et académiques. En tant que tel, les détails se rapportant aux tissus enrobage et de sectionnement rester un savoir-faire exclusif de la NSA. Partie 1: le traitement des animaux et de préparation des tissus Le cerveau d&#39;un singe adulte vervet (Cercopithecus aethiops) est utilisé pour le présent Protocole. Toutes les procédures sont menées en conformité avec le Conseil canadien de protection des animaux (CCPA) pour l&#39;utilisation et le soin des animaux en recherche biomédicale 1. <ol><li> Des animaux est profondément sédatés avec le chlorhydrate de kétamine (10 mg / kg, im), euthanasiés avec une overdose de pentobarbital sodique (25 mg / kg, iv) et perfusé transcardiaque avec PBS 0,1 M jusqu&#39;à ce que complètement exsangue. </li><li> Il est suivi par une solution de paraformaldéhyde 4% dans du PBS pendant 5 min (~ 1 litre). </li><li> Le cerveau est placé dans externalisée classés solutions de saccharose PBS tamponné (10, 20 et 30%, dans la séquence) contenant de l&#39;azoture de sodium à 0,02% et maintenu à 4 ˚ C jusqu&#39;à ce que le cerveau tombe au fond du conteneur. La solution est remplacé tous les quelques jours jusqu&#39;à ce cerveau subit le gel de sectionnement. </li><li> Le cerveau est envoyée aux Associés de NeuroScience (NSA; Knoxville, TN) pour être soumis à leurs propriétaires MultiBrain ™ enrobage et le gel de sectionnement de la technologie. </li><li> Sept points de repère d&#39;alignement (7) sont placés dans la matrice d&#39;inclusion afin que les articles peuvent être orientés correctement (c&#39;est-à-orientation gauche-droite) et re-aligné après traitement histologique est terminée. </li><li> Les blocs congelés est numériquement photographié à la face du bloc avant chaque section de série sont collectées à partir du bloc. Les images numériques ainsi que des sections à partir de ce cerveau peut ensuite être utilisé pour la reconstruction 3D des cartes anatomiques et histologiques, respectivement. </li><li> Serial flottant coupes coronales, sur une épaisseur de 50 microns par section, sont collectés par l&#39;ensemble du cerveau. </li></ol> Partie 2: traitement histologique Les articles sont choisis à un intervalle donné spatiales (par exemple, 500 um) et pour nos fins expérimentales ont été traitées avec les anticorps suivants: X fragile protéines retard mental (FMRP; Chemicon; Temecula, Californie), SMI32 (Sternberger monoclonaux Inc; Baltimore, MD), et NeuN (Chemicon; Temecula, Californie). FMRP est une protéine cytoplasmique qui abondamment présente dans les neurones du cerveau normal et porteuse de permutation 2. NeuN (noyaux neuronaux) reconnaît spécifiquement la NeuN DNA-binding protein spécifique des neurones, qui est présent dans la plupart des neurones et est distribué dans les noyaux des neurones, péricaryon et certains processus neuronaux proximale 3. SMI-32 est un anticorps monoclonal qui reconnaît l&#39;épitope non phosphorylée sur quatre protéines de neurofilaments. <ol><li> A un intervalle de 500 um, à environ 140 sections sont utilisés par des anticorps. </li><li> Toutes les incubations, lavages et étapes de coloration sont effectués avec une légère agitation à l&#39;aide des plats coloration spécialisés et paniers (HistoTools; Knoxville, TN). </li><li> Les articles sont d&#39;abord incubées dans une solution contenant 0,1 M PBS/0.3% de Triton X-100 (TX) / 5% de sérum normal enrouée (NHS) pendant 60 min, afin de réduire la liaison non spécifique des molécules d&#39;anticorps et une coloration de fond qui en résulte. </li><li> Les articles marqués d&#39;immunomarquage FMRP subir une étape supplémentaire de la récupération d&#39;antigène en utilisant la solution démasquage antigénique (Vector Labs; Burlingame, CA) conformément aux instructions du fournisseur. </li><li> Les articles sont ensuite incubés pendant la nuit et des jours différents dans leurs solutions d&#39;anticorps respectifs contenant 0,1 M PBS/0.3% TX / NHS 3% (avec un facteur de dilution de 1 / 5, 000 pour la FMRP et SMI-32 et 1 / 2, 000 pour les anticorps NeuN. </li><li> Le lendemain, les articles sont lavés pendant trois périodes de 10 min dans une solution de lavage (0,1 M PBS/0.3% TX) suivie d&#39;une incubation de 2 h à température ambiante dans biotinylé anti-souris anticorps secondaire soulevée dans cheval (Vector Labs, 1 / 1000 de dilution de 0,1 M PBS/0.3% NHS TX / 3%). </li><li> Après une autre série de trois lavages de 10 min, les sections sont placées dans une solution de complexe avidine-biotine peroxydase de raifort conjuguée (Vector Labs) pendant 1 h à température ambiante. </li><li> Enfin, après une autre série de lavages, les sections sont soumis pendant 10 min à un dia de nickel amélioréeminobenzidine (Ni-DAB) la réaction qui produit un bleu tache sombre dans les neurones immunoréactifs. </li><li> A l&#39;issue de la procédure histologique, les sections sont montées sur des lames de verre enduit de gélatine. </li><li> Toutes les sections sont séchés à l&#39;air pendant la nuit et lamelle, conformément aux procédures standard. </li></ol> Partie 3: Résultats du représentant: Cette méthode produit un profil d&#39;expression complète d&#39;une protéine cible d&#39;intérêt à travers un cerveau de singe entier. Ici, nous montrons que les coupes coronales représentatifs fournissent un instantané de la FMRP, NeuN et SMI32 expression dans le cerveau du singe même. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> Figure 1: Plateaux de coloration (A) et le panier (B) utilisé pour le traitement par lots à grande échelle de flottant des sections pour immunodétection. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> Figure 2: Représentant coupes coronales d&#39;un cerveau de singe vervet colorées pour FMRP (A), NeuN (B) et SMI32 (C) anticorps.

<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.

Il ya deux étapes essentielles de cette procédure qui font à grande échelle de détection des protéines dans un cerveau de singe toute possible. L&#39;un est le protocole enrobage et de sectionnement, qui reste le savoir-faire exclusif de la NSA. L&#39;autre est l&#39;utilisation de bacs de coloration et paniers fournis par HistoTools. Ce dernier permet une manipulation facile et rapide d&#39;un grand nombre (~ 40) sections à un moment donné. Il fournit également les moyens de traiter uniformément toutes les se...

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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

L&#39;immunohistochimie (IHC) est l&#39;une des techniques de laboratoire les plus utilisés pour la détection des protéines cibles<em&gt; In situ</em&gt;. Les questions concernant le profil d&#39;expression d&#39;une protéine cible dans le cerveau entier est relativement facile de répondre lorsque vous utilisez l&#39;IHC dans les cerveaux de petites, comme celles des rongeurs. Toutefois, répondre aux mêmes questions dans un grand cerveau et alambiquée, comme ceux des primates présente un certain nombre de défis. Nous présentons ici une approche systématique pour l&#39;immunodétection des protéines cibles dans un cerveau de singe adulte. Cette approche s&#39;appuie sur le tissu enrobage et de sectionnement méthodologie de NeuroScience Associés (NSA) ainsi que des outils développés spécifiquement pour les lots de la coloration flottant sections. Il en résulte une coloration uniforme d&#39;un ensemble de sections qui, à un intervalle particulier, représente l&#39;ensemble du cerveau. Les sections résultant tachés peuvent être soumis à une grande variété de procédures d&#39;analyse afin de mesurer les niveaux de protéines, la population de neurones exprimant une certaine protéine.

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

À grande échelle immunodétection des protéines cibles à travers le cerveau entier primates est possible en employant des tissus roman enrobage et de sectionnement des méthodes combinées avec l&#39;utilisation d&#39;appareils pour la coloration créative lot de plusieurs sections flottant à un moment donné.

Immunocoloration lots pour la détection de protéines à grande échelle dans le cerveau du singe entier

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

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Biology

12.6K vues.  Montreal Neurological Institute. À grande échelle immunodétection des protéines cibles à travers le cerveau entier primates est possible en employant des tissus roman enrobage et de sectionnement des méthodes combinées avec l'utilisation d'appareils pour la coloration créative lot de plusieurs sections flottant à un moment donné.

Vidéo: 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.

Grands écrans des banques métagénomiques

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

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Biologie

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.

La détection des protéines ubiquitination

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.

Purification de la M. magneticum Strain AMB-1 Protein magnétosome Associated 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.

Large Scale Zebrafish-Based In vivo Écran petites molécules

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.

Infinium Assay pour Grands génotypage SNP Applications

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.

Une stratégie pour sensibles à grande échelle quantitatives métabolomique

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.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

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: une méthode à grande échelle pour la quantification multiparamétrique de la croissance de la levure

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.

Identification de nouveaux régulateurs de la transpiration végétale par le dépistage thermique à grande échelle à 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 ...

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.

Immunomarquage de testicules de drosophiles de mont entier pour l’analyse confocale 3D de grands spermatocytes

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