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.

Alle tierischen Verfahren wurden von der Ethikkommission der Universität Mie genehmigt und wurden gemäß den japanischen Tierschutzverordnung &quot;Gesetz über die Wohlfahrt und Verwaltung von Animals &#39;(Ministerium für Umwelt, Japan) durchgeführt und mit den internationalen Richtlinien eingehalten werden. 1. Herstellung der Nadel HINWEIS: Alle Experimente wurden unter Vollnarkose durchgeführt, und alle Anstrengungen unternommen wurden, um das Leiden zu minimieren. Um Sterbehilfe, Fische wurden in einem Eis-Wasser-Bad (at ≤4 ° C 5 Teile Eis / 1 Teil Wasser) für ≥20 min eingetaucht. <ol><li> Bereiten Sie die Glas Mikrokapillare Nadeln durch Ziehen einer Glaskapillare 1,0-mm-Außendurchmesser mit einer Nadel puller (Abbildung 1A). </li><li> Schneiden Sie die Spitzen der Nadeln schräg mit einer feinen Schere (1B). 200 &amp; mgr; m (Abbildung 1c) - der ideale Spitzendurchmesser sollte etwa 100 sein. Wenn die Spitze diameter zu schmal ist, wird Blut nicht in die Nadel. </li><li> Aufzulösen Heparin in Kochsalzlösung auf eine Konzentration von 5 mg / ml. </li><li> Legen Sie eine vorgeschnittene Nadel im Objektivrevolver Ende einer Wasserstrahlpumpe Rohranordnung und halten Sie das Mundstück in den Mund, oder schließen Sie die Nadel in einer Glühbirne Spender. Tauchen Sie die Nadelspitze in der Heparin-Lösung und Heparinisierung die Nadel durch Saugen und Blasen durch die Lösung (Abbildung 1D und 1E). Hinweis: Das Ansaugrohr Montage für Zebrafisch Spermien Kryokonservierung 16 verwendet. Lange Gummischlauch kann kein Blut fliegen in den Mund zu stoppen. Setzen eines Filters in der Mitte der Röhre können die Gefahren zu vermeiden. </li><li> Bewahren Sie die heparinisierte Nadeln in einer 10 cm Petrischale und an der Luft trocknen für mindestens 1 Std. Eine große Anzahl von Nadeln im Voraus vorbereitet werden. </li></ol> 2. Anästhesie <ol><li> Bereiten Sie die Anästhesielösung in einem kleinen Kunststoffgehäuse durch Mischen von 200 ml Fischwasser mit 100 ul 2-Phenoxyethanol (2-PE). Endkonzentration der Anästhetika ist 500 ppm. VORSICHT! 2-PE hat eine schnelle Wirkung. </li><li> Entfernen gewünschte Anzahl von Fischen (AB-Stamm) aus dem Kreislauf-System. </li><li> Verwenden Sie ein Netz, um die Fische in die Anästhetika für 1 übertragen - 2 min (1F). Beobachten Sie die Fische nach und nach zu schwimmen, zu verbreiten, die Brustflossen horizontal, Keuchen und haben schnelle Operculum Bewegungen innerhalb 1 min. </li><li> Wie die Zeit vergeht, beobachten die Fische lagen auf dem Boden des Gehäuses und schließlich zu stoppen Schwimmen. Die chirurgische Ebene der Anästhesie wird erreicht, wenn der Fisch stoppt nach Luft zu schnappen und die Operculum Bewegungen sind langsam. An dieser Stelle ist Fisch bereit für die Blutentnahme. </li><li> Mit einem Schaumlöffel heben Sie den narkotisierten Fisch aus dem 2-PE und legen Sie sie vorsichtig auf ein Papiertuch mit den Anästhetika (1G) eingeweicht. Bedecken Sie den Kopf des Fisches mit weichem Tissue-Papier auch mit 2-PE-Lösung getränkt, um Augentrockenheit zu verhindern, und verwenden Sie ein anderes trockenes weiches Seidenpapier vorsichtigtrocknen die Körperoberfläche. </li></ol> 3. Blutentnahme <ol><li> Legen Sie eine heparinisierte Nadel im Objektivrevolver Ende des Ansaugrohr-Baugruppe (oder der Glühbirne Spender) und halten Sie die Mundstückende des Ansaugrohr Montage in den Mund. </li><li> Fassen Sie den Objektivrevolver Ende und die Nadel zusammen, und entfernen Sie vorsichtig die störenden Waagen mit der Nadelspitze. Führen Sie die Nadel in einem 30 - 45 ° Winkel in den Blutsammel Website. Durchstechen des Gastrointestinaltraktes (Figur 1H) vermeiden. Im Falle der Verwendung von Lampenspender, drücken Sie die Lampe mit Daumen und Mittelfinger, und blockieren die Öffnung an der Spitze der Lampe mit dem ersten Finger, und setzen Sie die Nadel wie oben beschrieben. Hinweis: Der Ort für die Blutentnahme ist entlang der Körperachse und hinter dem Anus in dem Bereich des dorsalen Aorta. Der dorsalen Aorta (DA) und die hintere Cardinalvene (PCV) nur ventral zur Wirbelsäule (Abbildung 2). </li><li> Fangen Sie an, die saugenMundstückende des Ansaugrohres Anordnung, wenn die Nadel zu berühren die Wirbelsäule zu spüren. Wenn Blut steigt nicht, bewegen Sie die Nadelspitze auf subtile Weise von Hand, um die Durchblutung zu fördern. Beachten Sie, dass, wenn Blut in die Nadel steigen, sofort aufhören zu zittern und saugen sanft (1I). Im Falle der Verwendung von Lampenspender, den Druck der Glühbirne, um das Blut zu saugen. </li><li> Beachten Sie das Blut langsam pulsatil in die Nadel steigen ohne Absaugung, die wegen der arterielle Blutdruck wahrscheinlich ist. Somit ist es nicht notwendig ist, zu saugen, wenn die Nadel die Arterie richtig eindringt. </li><li> Stoppen Absaugung nach dem geeigneten Volumen Blut gesammelt. Entfernen Sie die Nadel aus dem Fisch und drücken Sie die Einstichstelle mit weichem Tissue-Papier, um Blutungen zu stoppen. Beachten Sie die Blutung zu stoppen nach ca. 10 - 20 sec von Fingerdruck (Abbildung 1J). </li><li> Nachdem die Blutung gestoppt ist, den Fisch sofort wieder zurück zu übertragen, um eine cleein Warmwasser (~ 28 ° C) Tank. Helfen Sie dem Fisch vorsichtig wirbelnden Wasser zu den Kiemen, bis es anfängt zu schwimmen zu erholen. Hinweis: Bewahren Sie bis zu 5 postsampling Fisch in einem 2 l-Tank und verbinden Sie den Tank in den Kreislauf-System, um die Fische mit Sauerstoff zu versorgen. Zugabe von Antibiotika zum Fischwasser nicht erforderlich ist. Pflegen Sie den Fisch mit normaler Haltung und Fütterung. </li><li> Vertreibt das Blut von der Nadel auf eine saubere Fläche von einem Stück Parafilm (Abbildung 1 K). </li><li> Messen Blutzucker mit jeder kommerziellen Hand glucometer (Abbildung 1 l). Anmerkung: Die glucometer verwendet eine Glucose-Dehydrogenase-Flavinadenindinucleotid Elektrode und erfordert ein Probenvolumen von 0,6 &amp; mgr; l. <ol><li> Legen Sie einen Teststreifen vollständig in das Messgerät und direkt berühren Sie den Blutstropfen. Automatisch Beachten Sie die Blutentnahme in den Teststreifen und erhalten die Blutzucker 5 Sekunden auf der Anzeigefläche. Nehmen Sie das Ergebnis und entsorgen Sie den Teststreifen. Benutze einenneuen Teststreifen für jede Messung. </li></ol></li><li> (optionaler Schritt) Nehmen genaue Mengen von Blut durch eine Pipettiervorrichtung und übertragen das Blut in ein Mikrozentrifugenröhrchen für weitere Analysen (Hämoglobin, Triacylglycerin (TG), Gesamtcholesterin, etc.). Wenn nötig, verdünnen das Vollblut von einem Fisch mit Kochsalzlösung. Zentrifugieren der Blutproben für 3 min bei 680 · g bei RT und Ernte der Plasma. Übertragen des Plasmas in ein neues Röhrchen. An dieser Stelle ist es bereit, in biochemischen Analysen verwendet werden. </li></ol>

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The authors have nothing to disclose.

Wir stellen Ihnen hier eine detaillierte Protokoll zur seriellen Blutentnahme von erwachsenen Zebrafisch. Dieses Verfahren ist einfach durchzuführen und wir sie verwenden im Labor auf einer täglichen Basis. Das Blutsammelverfahren zum Einsetzen einer Glaskapillare Nadel in den Zebrafisch der dorsalen Aorta basiert. Während dieses Verfahrens ist es wichtig, vorsichtig zu sein, um nicht zu ablatieren die Wirbelsäule, weil sie das Kriterium für die Suche nach der dorsalen Aorta ist. Die Verringerung der Wirbelsäulenv...

Zebrafische sind gewinnt zunehmend an Popularität als ein wertvolles Modell der menschlichen Krankheiten, weil ihre Organe und Genetik ähnlich denen des Menschen 1,2 sind. Auf dem Gebiet der Entwicklungsbiologie, haben viele Studien gezeigt, dass die Zebrafisch und Menschen zeigen deutliche Ähnlichkeit in der Hämatopoese 3, Blutstillung 4,5 und Myelopoese 6. Erwachsenen Zebrafisch sind ebenfalls für die Untersuchung immunologischer 7, neurodegenerative 8 und Fettleibigkeit bedingte Krankheiten 9, weil dieses Modellorganismus Aktien gemeinsamen Wege mit denen in menschlichen Erkrankungen gestört verwendet. Für Übergewicht und Fettleibigkeit bedingte Krankheiten (Diabetes, Fettleber und nichtalkoholische Steatohepatitis und Atherosklerose), Zebrafisch-Blutzucker und Lipide Ebenen wurden gründlich in mehreren transgenen und ernährungsbedingte Fettleibigkeit Modelle 10-13 untersucht. Wiederholte Entnahme von Blut aus einzelnen Tiere Tiernutzung und Dezember zu reduzierenRease interindividuelle Unterschiede. Jedoch ist wiederholte Probennahme wegen ihrer relativ kleinen Blutvolumen und der Mangel an leicht zugänglichen Gefäßen technisch schwierig bei kleinen Tieren wie Zebrafisch. Verschiedene Methoden für die einmalige Blutentnahme von Zebrafisch entwickelt, obwohl diese Methoden haben ihre eigenen Nachteile, einschließlich Letalität assoziiert Gewebeschäden und begrenzte Blutvolumen. B. 1-5 &amp; mgr; l Blut von einem seitlichen Einschnitt von etwa 0,3 cm in der Länge in dem Bereich des dorsalen Aorta 5 geerntet werden. Enthauptung mit einer Schere durch Schneiden durch den Schultergürtel kann 5-10 &amp; mgr; l Blut 10 zu sammeln. Eine weitere praktische Blutentnahme Methode ist tail Ablation 14. Herzpunktion ist eine mögliche alternative Verfahren für wiederholte Blutentnahme aus dem gleichen Fisch, aber die sehr geringe Menge erhalten (etwa 50 nl) mit diesem Verfahren begrenzt die Anzahl der Analysen, die Leis sein kannMed. 11 Dementsprechend wird ein neues Protokoll benötigt, um wiederholter nicht-tödliche Blutabnahme, die zu kritischer Voraus erforderlich wäre dafür Organismus auf eine Standard-Modellorganismus für menschliche Krankheiten zu ermöglichen. Diese Technik würde zum Testen pharmakologische Reaktion, Entdeckung von Molekular Biomarker für die Diagnose, die Bestimmung der Prognose und die Überwachung von verschiedenen Krankheiten, wie Stoffwechselerkrankungen, degenerativen Erkrankungen und verschiedenen Arten von bösartigen Erkrankungen ermöglicht. Deshalb entwickelte eine minimal-invasive Verfahren zur Gewinnung von Blut von Zebrafischen in Reihe 15. Hier werden wir den Vorgang visuell nachweisen und ein detailliertes Protokoll für diese Technik. Unter Verwendung dieses Verfahrens wurden der normale Wert auf der Grundlage verschiedener Parameter, einschließlich Hämoglobin, Nüchternblutzucker und Lipide im Blut von gesunden erwachsenen Zebrafisch ausgewertet. Zusätzlich haben wir auch bewertet, ob diese Methode eignet sich für Studien, die serielle Proben durch m verlangenBERWACHUNG die zeitlichen Veränderungen der Blutzuckerwerte während der Überfütterung Experimenten.

<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

Wiederholte Blutentnahme ist eine der am häufigsten verwendeten Techniken bei Labortieren durchgeführt. Allerdings hat eine nicht-tödliche Protokoll für die Blutentnahme aus Zebrafisch nicht nachgewiesen. Die bisherigen Verfahren zur Blutentnahme aus Zebrafisch sind tödlich, wie laterale Inzision, Enthauptung und Schwanz Ablation. So haben wir eine neuartige &quot;wiederholte&quot; Blutabnahmeverfahren ein detailliertes Protokoll umreißt dieses Verfahren entwickelt, und heute hier. Dieses Verfahren ist minimal-invasiv und führt zu einer sehr geringen Sterblichkeit (2,3%) für Zebrafisch, wodurch wiederholte Entnahme von Blut aus dem gleichen Individuum. Das maximale Volumen der Blutabnahme ist abhängig vom Körpergewicht der Fische. Das Volumen für wiederholte Entnahme von Blut in Abständen sollte ≤0.4% des Körpergewichts pro Woche oder ≤1% alle 2 Wochen, was durch Messungen der Hämoglobin bewertet wurden sein. Zusätzlich Hämoglobin, Nüchternblutzucker, Plasma Triacylglycerol (TG) und c insgesamtholesterol Ebenen bei männlichen und weiblichen erwachsenen Zebrafisch wurden gemessen. Wir wendeten diese Methode auch, um die Fehlregulation des Glukosestoffwechsels in ernährungsbedingte Fettleibigkeit zu untersuchen. Dieses Blutsammelverfahren wird viele Anwendungen, einschließlich Glucose- und Fettstoffwechsels und hämatologischen Studien, die die Verwendung eines Zebrafischs als menschliche Krankheitsmodell Organismus erhöhen können.

Das Blutsammelverfahren bewirkt minimale Verletzung Zebrafisch (ein &lt;1 mm Punktion; Figur 1J) und ergibt eine sehr niedrige Mortalitätsrate von 2,3%. Wir untersuchten das maximale Volumen an Blut, die von einem einzelnen Fisch gesammelt und ausgewertet werden, die die Beziehung um sein Körpergewicht (Abbildung 3) werden könnte. Wir haben festgestellt, dass der maximale Blutvolumen gesammelt wurden linear mit dem Körpergewicht (R = 0,813) korreliert. Das größte Volumen von Blut ...

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.

Wiederholte Blutentnahme für Bluttests in Adult Zebrafisch

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 Aufrufe.  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.

Serie: 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.

Blutentnahme aus der amerikanischen 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 ...

Forschung

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.

Isolierung und Kultivierung von pulmonaler Endothelzellen aus neugeborenen Mäusen

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-mount Immunhistochemische Analyse für Embryonale Limb Haut Gefäßsystem: ein Modellsystem zur Vascular Branching Morphogenese in Embryo-Studie

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.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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.

In-vivo-Elektroporation von Morpholinos in das Regenerations Adult Zebrafisch Leitwerk

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.

Blutentnahme für Biochemische Untersuchungen an adulten Zebrafisch

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.

Gavaging Adult Zebrafisch

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.

Verfahren zum Screening und Validierung von Beständig Mutationen Vor-Inhibitoren

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.

Techniken für die Analyse der extrazellulären Vesikeln mittels Durchflusszytometrie

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).