This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

Toutes les procédures d&#39;animaux ont été approuvés par le comité d&#39;éthique de l&#39;Université de Mie, et ont été réalisées conformément à la réglementation de protection des animaux japonais &#39;loi sur la protection et la gestion des animaux »(ministère de l&#39;Environnement du Japon) et respectées aux lignes directrices internationales. 1. Préparation de l&#39;aiguille NOTE: Toutes les expériences ont été réalisées sous anesthésie, et tous les efforts ont été faits pour minimiser la souffrance. Pour l&#39;euthanasie, les poissons ont été immergés dans un bain d&#39;eau glacée (5 parties glace / 1 partie d&#39;eau à ≤4 ° C) pendant ≥20 min. <ol><li> Préparer les aiguilles de microcapillaires de verre en tirant un capillaire en verre de 1,0 mm de diamètre extérieur avec un extracteur d&#39;aiguille (Figure 1A). </li><li> Coupez les pointes des aiguilles à l&#39;aide de ciseaux fins oblique (figure 1B). Le diamètre de la pointe idéal devrait être d&#39;environ 100 - 200 um (figure 1C). Si la pointe diameter est trop étroite, le sang ne sera pas entrer dans l&#39;aiguille. </li><li> Dissoudre l&#39;héparine dans une solution saline à une concentration de 5 mg / ml. </li><li> Placer une aiguille prédécoupé à la fin de la tourelle d&#39;un ensemble de tube d&#39;aspiration et maintenez l&#39;embout buccal dans la bouche, ou de connecter l&#39;aiguille à un distributeur de l&#39;ampoule. Plonger la pointe de l&#39;aiguille dans la solution d&#39;héparine, et hépariner l&#39;aiguille par aspiration et de soufflage à travers la solution (figure 1D et 1E). Remarque: L&#39;ensemble de tube d&#39;aspiration a été utilisée pour la cryoconservation de sperme de poisson zèbre 16. Long tube de caoutchouc peut arrêter tout le sang battant dans la bouche. Définition d&#39;un filtre dans le milieu du tube peut éviter les dangers. </li><li> Stocker les aiguilles héparinés dans un plat de 10 cm de Petri et sécher à l&#39;air pendant au moins 1 h. Un grand nombre d&#39;aiguilles peut être préparé à l&#39;avance. </li></ol> 2. Anesthésie <ol><li> Préparer la solution anesthésique dans un petit boîtier en plastique en mélangeant 200 ml d&#39;eau de poissons avec 100 ul de 2-phénoxyéthanol (2-PE). La concentration finale des anesthésiques est de 500 ppm. ATTENTION! 2-PE a un effet rapide. </li><li> Retirer nombre désiré de poissons (souche AB) à partir du système de circulation. </li><li> Utilisez un filet de transférer le poisson dans les anesthésiques pour les 1 - 2 min (figure 1F). Observer les poissons nager progressivement, étaler les nageoires pectorales horizontalement, halètement, et avoir des mouvements de operculum rapide en 1 min. </li><li> Comme le temps passe, observer les poissons gisait sur le fond de l&#39;affaire et finalement arrêter la natation. Le plan chirurgical d&#39;anesthésie atteindra quand le poisson arrête à haleter et les mouvements sont lents opercule. À ce stade, le poisson est prêt pour la collecte de sang. </li><li> En utilisant une écumoire soulever le poisson anesthésié de la 2-PE et doucement le placer sur une serviette en papier imbibé d&#39;anesthésiques (figure 1G). Couvrir la tête du poisson avec du papier de soie douce aussi imbibé avec une solution 2-PE afin d&#39;éviter la sécheresse oculaire et utiliser un autre papier tissu doux et sec pour doucementsécher la surface du corps. </li></ol> 3. Collecte de sang <ol><li> Placer une aiguille héparine à la fin de la tourelle de l&#39;ensemble de tube d&#39;aspiration (ou le distributeur de l&#39;ampoule) et maintenir l&#39;extrémité de l&#39;embout de l&#39;ensemble de tube d&#39;aspiration dans la bouche. </li><li> Saisir l&#39;extrémité de nez et l&#39;aiguille ensemble, et retirez soigneusement les échelles interférant avec la pointe de l&#39;aiguille. Insérez l&#39;aiguille à un 30 - angle de 45 ° dans le site de collecte de sang. Éviter la perforation du tube digestif (figure 1H). En cas d&#39;utilisation de l&#39;ampoule distributeur, appuyer sur l&#39;ampoule avec le pouce et le majeur, et de bloquer le trou à la pointe de l&#39;ampoule avec le premier doigt, puis insérer l&#39;aiguille comme décrit ci-dessus. Remarque: Le site de collecte de sang est le long de l&#39;axe du corps et en arrière de l&#39;anus dans la région de l&#39;aorte dorsale. L&#39;aorte dorsale (DA) et la veine cardinale postérieure (PCV) sont juste ventrale à la colonne vertébrale (figure 2). </li><li> Commencer à sucer leembout buccal extrémité de l&#39;ensemble de tube d&#39;aspiration lorsque l&#39;aiguille se fait sentir touchant la colonne vertébrale. Si le sang ne se lève pas, déplacer la pointe de l&#39;aiguille subtilement à la main pour favoriser la circulation sanguine. Notez qu&#39;une fois que le sang est à la hausse dans l&#39;aiguille, arrêter immédiatement secousses et sucer doucement (Figure 1I). En cas d&#39;utilisation de l&#39;ampoule distributeur, relâcher la pression de l&#39;ampoule pour aspirer le sang. </li><li> Observez le sang va augmenter lentement dans l&#39;aiguille d&#39;une manière pulsatile sans aspiration, qui est probablement en raison de la pression artérielle. Ainsi, il est inutile d&#39;aspirer si l&#39;aiguille pénètre dans l&#39;artère correctement. </li><li> Arrêtez aspiration après le volume approprié de sang est recueilli. Retirez l&#39;aiguille du poisson et appuyez sur le site de ponction à l&#39;aide de papier tissu doux pour arrêter les saignements. Observez l&#39;arrêt des saignements après environ 10 - 20 sec de la pression du doigt (figure 1J). </li><li> Après le saignement est arrêté, transférer immédiatement le poisson à un articleun-eau chaude (~ 28 ° C) du réservoir. Aidez le poisson à récupérer en remuant doucement l&#39;eau vers les branchies jusqu&#39;à ce qu&#39;il commence à nager. Remarque: Gardez jusqu&#39;à 5 poissons postsampling dans un réservoir de 2 L et raccorder le réservoir au système de circulation pour oxygéner le poisson. L&#39;addition d&#39;antibiotiques à l&#39;eau des poissons est pas nécessaire. Maintenir le poisson avec le logement et l&#39;alimentation normale. </li><li> Expulser le sang de l&#39;aiguille sur une surface propre d&#39;un morceau de parafilm (figure 1K). </li><li> Mesurer la glycémie en utilisant toute glucomètre portatif commerciale (figure 1L). Remarque: Le glucomètre utilise une déshydrogénase-flavine-adénine-dinucléotide électrode de glucose et nécessite un volume de 0,6 ul d&#39;échantillon. <ol><li> Insérez une bandelette de test complètement dans le compteur et directement toucher la goutte de sang. Observez le tirage au sort de sang dans la bande de test automatiquement et obtenir le résultat de la glycémie 5 sec sur la zone d&#39;affichage. Enregistrez le résultat et jetez la bandelette de test. Utiliser unnouvelle bande pour chaque mesure d&#39;essai. </li></ol></li><li> (étape optionnelle) Prendre des quantités précises de sang par un pipetage et transférer le sang dans un tube à centrifuger pour d&#39;autres analyses (hémoglobine, triacylglycérols (TG), le cholestérol total, etc.). Si nécessaire, diluer le sang total d&#39;un poisson avec une solution saline. Centrifuger les échantillons de sang pendant 3 min à 680 x g à la température ambiante et récolter le plasma. Transférer le plasma dans un nouveau tube. À ce stade, il est prêt à être utilisé dans des analyses biochimiques. </li></ol>

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The authors have nothing to disclose.

Nous présentons ici un protocole détaillé pour l&#39;obtention de série sang de poisson zèbre adulte. Cette méthode est simple à réaliser et nous l&#39;utilisons dans le laboratoire sur une base quotidienne. Cette méthode de collecte de sang est basée sur insérant une aiguille capillaire en verre dans l&#39;aorte dorsale du poisson zèbre. Au cours de cette procédure, il est essentiel de veiller à ne pas pratiquer l&#39;ablation de la colonne vertébrale, car il est le critère pour la recherche de l&#39;ao...

Zebrafish gagnent en popularité comme un modèle précieux de maladies humaines parce que leurs organes et la génétique sont similaires à ceux des humains 1,2. Dans le domaine de la biologie du développement, de nombreuses études ont démontré que le poisson zèbre et de montrer humaine marqués similitude dans l&#39;hématopoïèse 3, hémostase 4,5 et 6 myélopoïèse. Poisson zèbre adultes sont également utilisés pour l&#39;étude immunologique 7, 8 et les maladies neurodégénératives liées à l&#39;obésité 9 car cet organisme modèle actions voies communes avec ceux perturbé dans les maladies humaines. Pour l&#39;obésité et les maladies liées à l&#39;obésité (diabète, la stéatose hépatique non alcoolique et la stéatohépatite et athérosclérose), le poisson-zèbre niveaux de lipides et de glucose dans le sang ont été examinées à fond dans plusieurs modèles transgéniques et l&#39;obésité induite par l&#39;alimentation 10-13. Répétée prélèvement de sang d&#39;animaux individuels permettra de réduire l&#39;utilisation des animaux et diminuerRease différences interindividuelles. Cependant, la collecte de l&#39;échantillon répété est techniquement difficile chez les petits animaux tels que le poisson-zèbre à cause de leur volume de sang relativement faible et le manque de récipients facilement accessibles. Plusieurs méthodes de collecte de sang ponctuelle de poisson zèbre ont été développés, bien que ces méthodes ont leurs propres inconvénients, y compris la létalité, lésions tissulaires associées et le volume sanguin limitée. Par exemple, 1 à 5 ul de sang peut être récolté à partir d&#39;une incision latérale d&#39;approximativement 0,3 cm de longueur dans la zone de l&#39;aorte dorsale 5. Décapitation avec des ciseaux en coupant à travers la ceinture scapulaire peut recueillir le sang de 5-10 ul 10. Une autre méthode de prélèvement de sang est commode queue ablation 14. Ponction cardiaque est une méthode alternative potentielle pour la collecte de sang répétées du même poisson, mais la très petite quantité obtenue (environ 50 nl) avec cette procédure limite le nombre d&#39;analyses qui peuvent être performed 11. En conséquence, un nouveau protocole est nécessaire pour permettre un échantillonnage répété de sang non-létale, ce qui serait une avancée critique nécessaire pour cet organisme d&#39;être un organisme modèle standard pour les maladies humaines. Cette technique permettrait d&#39;essai réponse pharmacologique, la découverte de marqueurs biologiques moléculaires pour le diagnostic, la détermination du pronostic et la surveillance de diverses maladies telles que les maladies métaboliques, des maladies dégénératives et plusieurs types de tumeurs malignes. Nous avons donc développé une méthode minimalement invasive pour obtenir le sang de poisson zèbre en série 15. Ici, nous démontrons la procédure visuellement et fournissons un protocole détaillé pour cette technique. En utilisant cette méthode, la valeur normale sur la base de différents paramètres, dont l&#39;hémoglobine, la glycémie à jeun, et de lipides dans le sang du poisson zèbre adultes en bonne santé ont été évalués. En outre, nous avons également évalué si cette méthode est adaptée pour les études qui nécessitent des échantillons en série par murveillance les changements temporels dans les niveaux de glucose sanguin au cours des expériences suralimentation.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass capillaries with filament</td>
			<td>Narishige</td>
			<td>GD-1</td>
			<td>1.0-mm-outer-diameter.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>To produce the needls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>081-00136</td>
			<td>For heparinization</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Drummond</td>
			<td>2-040-000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Bulb dispenser</td>
			<td>Drummond</td>
			<td>1-000-9000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-12075</td>
			<td>For anesthetizing the fish</td>
		</tr>
		<tr>
			<td>DRI-CHEM3500V</td>
			<td>Fujifilm</td>
			<td>-</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>DRI-CHEM Slides</td>
			<td>Fujifilm</td>
			<td>Hb-WII</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>Glutest Neo Super</td>
			<td>Sanwa Kagaku Kenkyusho</td>
			<td>-</td>
			<td>For bood glucose measurement</td>
		</tr>
		<tr>
			<td>Wako L-type TG kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>464-44201</td>
			<td>For TG measurement</td>
		</tr>
		<tr>
			<td>Wako L-type CHO kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>460-44301</td>
			<td>For total cholesterol measurement</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan Packaging</td>
			<td>PM996</td>
			<td>To expel the blood on</td>
		</tr>
	</tbody>
</table>

repeated blood collection for blood tests in adult zebrafish

Collecte de sang répétées est une des techniques les plus courantes effectuées sur des animaux de laboratoire. Cependant, un protocole non létal pour le prélèvement de sang à partir du poisson zèbre n&#39;a pas été établie. Les méthodes précédentes pour la collecte de sang de poisson zèbre sont mortelles, comme incision latérale, la décapitation et la queue ablation. Ainsi, nous avons développé une nouvelle méthode &quot;répétée&quot; de collecte de sang, et présente ici un protocole détaillé décrivant cette procédure. Cette méthode est peu invasive et se traduit par un taux de mortalité très faible (2,3%) pour le poisson-zèbre, permettant ainsi répété le prélèvement de sang à partir du même individu. Le volume maximal de prélèvement de sang est fonction de poids du corps du poisson. Le volume pour un échantillonnage répété de sang à des intervalles doit être ≤0.4% du poids du corps de chaque semaine ou ≤1% toutes les 2 semaines, qui ont été évalués par des mesures de l&#39;hémoglobine dans le sang. En outre, l&#39;hémoglobine, la glycémie à jeun, le plasma triacylglycérols (TG) et c totaleniveaux de holesterol chez le poisson zèbre adultes mâles et femelles ont été mesurés. Nous avons également appliqué cette méthode pour étudier la dysrégulation du métabolisme du glucose dans l&#39;obésité induite par l&#39;alimentation. Cette méthode de collecte de sang permet de nombreuses applications, y compris le glucose et le métabolisme des lipides et des études hématologiques, ce qui augmentera l&#39;utilisation du poisson zèbre comme organisme modèle de maladie humaine.

Cette méthode de collecte de sang provoque des lésions minimes à poisson zèbre (un &lt;1 mm perforation; Figure 1J) et donne un taux de mortalité très faible de 2,3%. Nous avons examiné le volume maximum de sang qui pourraient être recueillies à partir d&#39;un seul poisson et évalué la relation à son poids de corps (Figure 3). Nous avons constaté que le volume de sang recueilli a été maximale linéairement corrélée avec le poids corporel (R = 0,813). Le plus grand volu...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Collecte de sang répétées pour des tests sanguins dans le poisson zèbre adulte

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.5K vues.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Vidéo: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Collecte de sang du crabe américaine Horseshoe, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Recherche

Biologie

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolement et culture de cellules endothéliales pulmonaires des souris néonatales

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-Mont analyse immunohistochimique à la vascularisation cutanée Limb embryonnaires: un modèle pour l&#39;étude vasculaire morphogenèse de branchement de l&#39;embryon

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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 Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En électroporation in vivo de morpholinos dans la régénération des adultes Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Collecte de sang pour analyse biochimique chez le poisson zèbre adulte

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavage poisson zèbre pour adultes

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Un procédé de criblage et de validation des mutations de résistance aux inhibiteurs de kinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Techniques pour l&#39;analyse des vésicules extracellulaire par cytométrie en flux

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).