Les méthodes de fécondation in vitro et diploïdes gynogénétiques ont été développés pour le poisson-zèbre par Charline Walker dans le laboratoire de George Streisinger à l&#39;Université de l&#39;Oregon dans les années 1970 et 1980. La méthode décrite ici est inchangé par rapport à des protocoles Charline, qui sont disponibles dans leur intégralité dans le Livre Zebrafish 3. Les méthodes de fécondation in vitro et diploïdes gynogénétiques ont été développés pour le poisson-zèbre par Charline Walker dans le laboratoire de George Streisinger à l&#39;Université de l&#39;Oregon dans les années 1970 et 1980. La méthode décrite ici est inchangé par rapport à des protocoles Charline, qui sont disponibles dans leur intégralité dans le Livre Zebrafish 3.

Aperçu de la pression de début Remarque: Les recettes pour la plupart des réactifs utilisés dans ce protocole tel que tricaïne, poissons d&#39;eau salée et Hanks sont disponibles en ligne dans le chapitre 10 du Livre Zebrafish 3 ( <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ) <ol><li> Produire des embryons par fécondation in vitro en utilisant le sperme UV inactivé. </li><li> Transférer immédiatement les oeufs fécondés dans un flacon en verre (avec un plafond pression en plastique) rempli à ras bord avec de l&#39;eau d&#39;œuf. Couvrir l&#39;extrémité ouverte du flacon avec une mince feuille de latex de caoutchouc et enclenchez le haut (dans lequel un trou a été coupé) sur le flacon sur le caoutchouc. </li><li> Placer le flacon dans une presse hydraulique et à 1,4 min après la fécondation augmenter la pression à 8000 psi (Notez que les presses nombreuses sont calibrés pour lire la pression exercée sur un piston de diamètre de 2 pouces, et les lectures doivent être convertis en psi) </li><li> Début à 6 min après la fécondation, libèrent graduellement la pression sur une période de 1 min. </li></ol> Détail procédure de la pression au début <ol><li> Jour avant l&#39;expérience <ol style="list-style-type:lower-alpha;"><li> En fin d&#39;après midi, les mâles et les femelles séparés pour être pressé. Tout vieux mâle peut être utilisé car il ne sera pas contribué du matériel génétique. Les femelles doivent chercher de grandes et de graisse dans le ventre. Les mâles doivent chercher jaune et Spry. </li><li> Tenez femelles et les mâles séparément pour la nuit dans des réservoirs où ils peuvent être facilement accessibles dans la matinée. </li></ol></li><li> Matin d&#39;expérience <ol style="list-style-type:lower-alpha;"><li> Préparer la solution de Hanks, ajouter le bicarbonate de sodium. </li><li> Apportez à la facilité de poissons: seau à glace, Hanks, tricaïne, minuterie, les tubes de collecte de sperme. </li><li> Diluer dans de l&#39;eau des poissons tricaïne </li></ol></li><li> Recueillir le sperme <ol style="list-style-type:lower-alpha;"><li> Préparer un flacon transparent d&#39;une solution de Hanks sur la glace. Mesure ~ 50 ul par des hommes à être pressé. </li><li> Anesthetize 3 ou 4 mâles à la fois par immersion dans tricaïne. Ils peuvent être sorti de l&#39;anesthésie avec une cuillère en plastique. </li><li> Rincer à l&#39;eau les poissons et épongez «humide-sèche&quot; sur une serviette en papier (très important: l&#39;eau actionne des spermatozoïdes). </li><li> Mont de poisson dans un porte-mousse sous un stéréomicroscope à faible grossissement avec épi-illumination. Séparez les nageoires pelviennes avec une pince et la position d&#39;un microcapillaire à l&#39;ouverture du cloaque. </li><li> Course sur les côtés du poisson doucement mais fermement avec douceur (Millipore) forceps. </li><li> Comme le sperme laiteux sortir du pore génital, le recueillir dans le microcapillaire en utilisant une aspiration douce. </li><li> Piscine du sperme de plusieurs mâles dans une solution saline glacée de Hank. Le sperme des mâles de 50 à 10 est suffisant pour la fécondation de plusieurs centaines d&#39;oeufs. Le sperme dans un froid Hanks va continuer à fertiliser les œufs efficacement pendant plusieurs heures voire plusieurs jours. &quot;Eyeball&quot; la concentration des spermatozoïdes, la collecte assez pour faire une suspension trouble. </li></ol></li><li> UV inactiver les spermatozoïdes <ol style="list-style-type:lower-alpha;"><li> Remplissez le fond d&#39;un plat en verre de Pétri avec de la glace. </li><li> Mettez un verre de montre sur le dessus de la glace dans le plat. </li><li> En utilisant une pipette Pasteur, transférer le sperme de l&#39;éprouvette sur le verre de montre. Soyez sûr d&#39;obtenir autant de sperme dans la pipette que possible sans prendre toutes les bulles d&#39;air avec elle. Aussi, ne pas la bulle du sperme dans le verre de montre que vous êtes les expulser de la pipette. Expulser des spermatozoïdes dans une fine couche sur le verre de montre. JETER cette pipette. </li><li> Placez la boîte de Pétri dans une UV réticulant </li><li> Allumez le réticulant pendant 2 minutes </li><li> Utiliser une pipette propre, enlever le sperme du verre de montre et de le transférer dans un tube Eppendorf a marqué sperme UV et placer sur la glace. </li></ol></li><li> Collecte des oeufs et de la fécondation in vitro <ol style="list-style-type:lower-alpha;"><li> Anesthetize trois ou quatre femelles à la fois dans tricaïne. </li><li> Rincer à l&#39;eau les poissons et épongez «humide-sèche&quot; sur une serviette en papier. L&#39;excès d&#39;eau va gonfler les oeufs et empêcher la fécondation. </li><li> Placer la femelle dans un 35 mm en plastique et des boîtes de Pétri avec un linge humide (pas mouillé) doigts, appuyez doucement mais fermement sur le ventre. Si elle est prête à pondre, ils sortiront facilement. </li><li> Rassemblez les oeufs avec une spatule et retourner à la femelle de l&#39;eau. Bonne oeufs sont un aspect jaunâtre, couleur translucide, tandis que les œufs qui sont restés dans la femelle trop long sont blancs et aqueux (voir ci-dessous). Pour assurer une bonne obtenir des œufs, les recueillir durant les 2 premières heures après les lumières s&#39;allument dans l&#39;établissement de poissons. Gardez les oeufs couverts pour éviter le dessèchement. </li><li> Ajouter 30 à 50 pi de la suspension de spermatozoïdes UV pour les œufs, mélanger délicatement avec la pointe de pipette. </li><li> ImmédiateLy ajouter environ 1 ml d&#39;eau les poissons et démarrage de la minuterie. Oeufs de gauche à se développer à ce point sera haploïdes. </li></ol></li><li> . Pression Early Remarque: les étapes AG doit être accomplie dans les 90 secondes de la fécondation! <ol style="list-style-type:lower-alpha;"><li> Démarrer minuterie au moment où les œufs sont activés par l&#39;ajout d&#39;eau </li><li> Attendez quelques instants et ajouter plus d&#39;eau, tourbillon les oeufs au centre du plat. </li><li> En utilisant une teneur de coupure et poli pipette Pasteur, transférer les oeufs fécondés pour les flacons de pression. </li><li> Si nécessaire, ajoutez de l&#39;eau plus d&#39;œufs sur les flacons de sorte qu&#39;ils sont presque débordante, laissant une perle d&#39;eau à la lèvre des flacons. </li><li> Placez un bouchon en caoutchouc sur la perle d&#39;eau et fermer le couvercle en plastique sur le caoutchouc. </li><li> Placer le flacon (s) à l&#39;envers dans le cylindre de pression, en ajoutant de l&#39;eau plus d&#39;œufs pour remplir le cylindre. Mettre le dessus sur le cylindre en toute sécurité. </li><li> Placer le cylindre dans la presse avec le piston et appliquer £ 8,000 / m². po </li><li> Laisser le cylindre dans la presse jusqu&#39;à 6 min après le moment de l&#39;activation (pour un temps total de 4,5 min de pression). </li><li> A 6 minutes après l&#39;activation, retirer le cylindre de la presse, et sortir les flacons du cylindre. Séchez soigneusement les flacons pour éviter le refroidissement de l&#39;embryon. </li><li> Réinitialiser le cylindre et le remplir avec de l&#39;eau d&#39;œuf. </li><li> Préparer les flacons prochaine partiellement les remplir avec de l&#39;eau d&#39;œuf, au besoin. </li><li> Répétez l&#39;opération pour tous les lots d&#39;œufs. </li></ol></li></ol> NOTE: Le cylindre peut contenir jusqu&#39;à 3 flacons à la fois, de sorte que vous voudrez peut-être de retarder la fécondation des œufs de quelques minutes pour voir si d&#39;autres femmes déjà dans la tricaïne donnent des oeufs. Cela aidera à accélérer les choses en traitant plusieurs lots en même temps. Aussi, n&#39;oubliez pas une fois la presse est utilisée ne mettez pas plus de poissons dans la tricaïne jusqu&#39;à ce que le traitement est terminé. Permettant aux poissons de rester dans l&#39;anesthésie trop longtemps peut les tuer. Deuxièmement, si vous pressez le poisson alors que la presse est utilisée, et obtenir des œufs, ils peuvent être trop sec pour être viable au moment où la presse peut être utilisée de nouveau.

<ol>
	<li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112 (2), 311-311 (1986).</li><li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-293 (1981).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Johnson, S. L., Africa, D., Horne, S., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-tetrad+analysis+in+zebrafish:+mapping+the+ros+mutation+and+the+centromere+of+linkage+group+I.">Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.</a> Genetics. 139 (4), 1727-1727 (1995).</li><li>Beattie, C. E., Raible, D. W., Henion, P. D., Eisen, 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=Early+pressure+screens.">Early pressure screens.</a> Methods Cell Biol. 237 (71), 2575-25 (1999).</li><li>Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-linkage+analysis+and+consolidation+of+the+zebrafish+genetic+map.">Centromere-linkage analysis and consolidation of the zebrafish genetic map.</a> Genetics. 142 (4), 1277-1277 (1996).</li><li>Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutants+with+disrupted+early+T-cell+and+thymus+development+identified+in+early+pressure+screen.">Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.</a> Dev Dyn. 237 (9), 2575-2575 (1999).</li></ol>

Il est important de s&#39;assurer que les embryons produits par cette méthode sont diploïdes gynogénétiques vrai. Comme contrôles, de générer une couvée d&#39;oeufs diploïdes normales (en gardant de côté certaines de sperme sans UV-inactivation) et un embrayage d&#39;embryons haploïdes (en gardant de côté une couvée d&#39;oeufs fécondés avec le sperme UV sans passer par la procédure du PE). Au 1er embryons après la fécondation haploïde jour ont un axe corps court, le cerveau et la morphologie irrég...

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making gynogenetic diploid zebrafish by early pressure

Hétérozygotie chez les eucaryotes diploïdes fait souvent des études génétiques lourdes. Les méthodes qui produisent des descendants viables diploïdes homozygotes directement à partir de femelles hétérozygotes permettent F1 femelles mutagénisées à être projeté directement mutations délétères dans un écran accélérée avant génétique. Streisinger et al.<sup&gt; 1,2</sup&gt; Décrit des méthodes pour rendre le poisson-zèbre gynogénétiques diploïde (homozygote) en activant œufs de poisson zèbre avec la lumière ultraviolette inactivé sperme et la prévention soit la deuxième ou la méiose de la première division cellulaire zygotique utilisant des traitements physiques (chaleur ou la pression) que les microtubules deploymerize. La &quot;pression précoce» (EP) bloque la méthode II de la méiose, qui survient peu après la fécondation. La méthode EP produit un pourcentage élevé d&#39;embryons viables qui peuvent se développer à des adultes fertiles des deux sexes. La méthode génère des embryons qui sont homozygotes à tous les loci, sauf ceux qui ont été séparés de leur centromère par recombinaison durant la méiose I. mutations homozygotes sont détectés dans les embrayages PE entre 50% pour les loci centromérique et moins de 1% pour les loci télomériques. Cette méthode est reproduite textuellement du livre Zebrafish<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Making Gynogenetic Diploid Zebrafish by Early Pressure at JoVE.com

Making Gynogenetic Diploid Zebrafish by Early Pressure

C&#39;est une méthode pour générer des embryons de poisson zèbre gynogénétiques diploïde (embryons dont la seule contribution génétique vient de la mère) en bloquant la seconde division méiotique immédiatement après la fécondation avec la lumière ultraviolette inactivé sperme. EP embryons ne sont pas entièrement homozygotes due à la recombinaison lors de la première division méiotique, cependant elles sont homozygotes à tous les loci qui n&#39;ont pas été séparés de leur centromère par recombinaison.

Faire Zebrafish diploïdes gynogénétiques par Pression Early

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

making-gynogenetic-diploid-zebrafish-by-early-pressure

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Biology

13.4K vues.  University of Oregon. C'est une méthode pour générer des embryons de poisson zèbre gynogénétiques diploïde (embryons dont la seule contribution génétique vient de la mère) en bloquant la seconde division méiotique immédiatement après la fécondation avec la lumière ultraviolette inactivé sperme. EP embryons ne sont pas entièrement homozygotes due à la recombinaison lors de la première division méiotique, cependant elles sont homozygotes à tous les loci qui n'ont pas...

Vidéo: Making Gynogenetic Diploid Zebrafish by Early Pressure

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

En direct l&#39;imagerie du cerveau de poisson zèbre embryonnaires par microscopie confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Recherche

Biologie

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Banques de cerveaux: Tirer le meilleur parti de vos spécimens de recherche

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

Faire jouer IRM enfant - Protocole neuroimagerie pédiatrique, lignes directrices et la procédure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

Génération embryons de poissons zèbres chimériques par transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Pression atmosphérique imagerie moléculaire des tissus biologiques et biofilms par spectrométrie de masse LAESI

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analyse directe des cellules isolées par spectrométrie de masse à la pression atmosphérique

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glycanes imagerie dans embryons de poissons zèbres par marquage métabolique et Chimie Cliquez Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Production de haploïdes embryons de poisson zèbre par<em&gt; In Vitro</em&gt; Engrais

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...