The authors have nothing to disclose.

Alle Verfahren werden nach den in &quot;Principles of Laboratory Animal Care&quot; beschriebenen Richtlinien durchgeführt (National Institutes of Health Veröffentlichung nicht. 85-23, überarbeitet 1985) und der genehmigte Rosalind Franklin Universität Institutional Animal Care und Verwenden Ausschuss Tier Protokoll 08-19. 1. Gentamicin-Induktion von Haarzell Nekrose <ol><li> Vorbereitung Gentamicinsulfat in normaler Salzlösung in einer Endkonzentration von 0,004% (4,32 mM). </li><li> Zeigen erwachsenen Fischen (D. rerio, 4-6 Monate alt) in einem Behälter, der die 0,004% (4,32 mM), Gentamicin-Lösung. Einem Behälter verwendet werden, aber wir von einem Pharmacal Wasser-System, das in 7 breit, 6 in hohen und lang ist in 7 mit einem Fischbehälter. Stellen Sie den Behälter mit Fisch in einem Brutschrank bei 28 ° C für 24 h eingestellt. Stellen Sie das gesamte Fluidvolumen in dem Tank auf einem ausreichenden Niveau, um Fische in einem lebensfähigen Zustand für den Zeitraum von 24 Std. erhalten. Hinweis: Die Belüftung des Gentamicin-Flüssigkeit nicht necessar Falls genügend Volumen für die Anzahl der zu behandelnden Fisch verwendet. </li></ol> 2. Vital Färbung der Haarzellen <ol><li> Vorbereitung einer 0,08% Konzentration (in normaler Kochsalzlösung) des Fluoreszenzvitalfarbstoff [4-4-Diethylaminostyryl)-N-methylpyridiniumiodid (485 nm Anregung und 603 nm, λ Emission λ in Methanol) von einer Arbeitsstammlösung von 15 mg / ml in Ethanol. </li><li> Um festzustellen, ob die Behandlung wirksam war Gentamicin eine Teilmenge der Kontrolle und Gentamicin behandelten Fische werden sofort, indem sie Fische in der Vertiefung einer 6-Well-Kulturplatte, die den Vitalfarbstoff angefärbt. Verwenden Sie eine ausreichende Anzahl von Fischen (und Kulturplatten für statistische Signifikanz erreicht werden benötigt. Basierend auf der Prüferin Geschwindigkeit von Neuromasten zählen, den Fisch in den Platten gestaffelt im Laufe der Zeit, so dass die Fische nicht mehr als 75 min angefärbt wie in Schritt 2.3 beschrieben. </li><li> Legen Sie die Platten aus Schritt 2.2 in einer Tischschublade von dem Fluoreszenzmikroskopzur Untersuchung von gefärbten Neuromasten verwendet werden. Schalten Sie die Raumbeleuchtung auf Löschung der Vitalfarbstoff über die 1 Stunde Färbung Zeitraum bei Raumtemperatur zu verhindern. </li><li> Bereiten Sie sowohl Farbstoff Wash-out-und Anästhesiewassertanks. Dye Wash-out-Wasser ist normal, Wasser und Fisch für Narkose Wasser, fügen Sie genügend 2-Phenoxyethanol, so dass eine 1:1000 Verdünnung in normalen Fischwasser erreicht. </li><li> Zeigen Fisch über normale Fische, um überschüssiges Wasser spülen Vitalfarbstoff, und fahren Sie zur Beobachtung der Vitalfarbstoff angefärbt Fisch Schritt 3.1. </li><li> Um die Regeneration der Neuromasten zu untersuchen, zu übertragen Gentamicin-behandelte Fische, die in normalen Fischwasser in einen Inkubator für zwischen 8-16 h bei 28 ° C gewaschen </li><li> Zu verschiedenen Zeitpunkten zwischen 8-16 h werden die Fische aus dem Inkubator entfernt, gewaschen und gefärbt, wie in den Schritten 2.1-2.4 angegeben. Gehen Sie zur Beobachtung der Vitalfarbstoff angefärbt Fisch Schritt 3.1. </li></ol> 3. Anästhesie Fisch und Fluorescent Auszählung der Neuromasten <ol><li&gt; Tupfen Sie jeden Fisch auf einem Papiertuch, um überschüssige Flüssigkeit zu entfernen und dann legen Sie sie auf einem feuchten Stück Filterpapier, das auf dem Deckel einer Petrischale aus Kunststoff zentriert ist. </li><li> Setzen Sie den Deckel auf der Bühne eines Fluoreszenzstereomikroskop, um ein digitales Bild der Vitalfarbstoff gefärbten Neuromasten der Mitte der Körper Stiche erhalten. </li><li> Verwenden Sie eine Digitalkamera auf dem Fluoreszenzstereomikroskop gelegt gesetzt eine Vergrößerung von 2X, um Bilder für die anschließende quantitative Analyse zu erfassen. Hinweis: Die Vergrößerungseinstellung des Stereomikroskops kann auf die Marke von Mikroskop verwendet werden, hängen, aber die Einstellung ist die einfache Anzeige und Zählung der einzelnen Neuromasten in der Mitte der Körper Stiche zu ermöglichen. </li><li> Bestimmung der Regenerationsmenge durch Zählen der Anzahl sichtbarer Neuromasten in den vier benannten Maschen auf der untersten Bauchseite des Fisches proximal der rechten Brustflosse (siehe Abbildung 1). Für die statistische Analyse verwenden Sie eine geeignete Test wie ANOVA or des Student-T-Test. Experimente sollte ein Minimum von 5 Fische pro Zeitpunkt nutzen und alle Experimente sollte mindestens 3x wiederholt werden. </li><li> Basierend auf der Neuromasten Regenerationszeit-Kurve (siehe Abbildung 3), zählen Neuromasten zwischen 8-16 Stunden nach Gentamicin-wash-out innerhalb der linearen Phase der Regeneration Kurve. Hinweis: Die Verwendung der linearen Zeitphase ermöglicht die richtige quantitative Analyse zwischen der Kontroll-und Versuchsgruppen. </li></ol> 4. Fluorescent Zählen einzelner Haarzellen zur Gewinnung Höhere Auflösung des Quantitative Analyse, wenn die Neuromasten Analyse statistisch nicht signifikant <ol><li> Wenn die quantitative Analyse auf der Ebene der Neuromasten nicht signifikant ist, kann die Analyse auf der Ebene der einzelnen Haarzellen auch verwendet werden, um eine höhere Auflösung zu erhalten. Wählen Fisch zu einem bestimmten Zeitpunkt nach Gentamicin-wash-out (Zeitpunkt auf der Basis der früheren Studien Neuromasten), Vitalfarbstoff stain den Fisch in Protokoll 2 beschrieben, und einschläfern den Fisch mit 2-Phenoxyethanol in einer Verdünnung von 1:500 für 1-5 min dann. </li><li> In gedämpftem Licht zu Abschrecken zu vermeiden, stellen vier Einschnitte, so dass ein Quadrat Hautlappen Zubereitung wird wie folgt hergestellt. Einen Einschnitt entlang der oberen Rippen der Fische, bis es mit den Afterflossen ausgerichtet ist, dann machen Sie einen Schnitt über den Bauch und schließlich, stellen zwei vertikale Schnitte auf jeder Seite dieser Einschnitte, so das Quadrat Hautlappen wird erstellt. Hinweis: Diese Vorbereitung der Haut wird die Mitte Körper Stiche in den Neuromasten Experimente verwendet zu integrieren. </li><li> Legen Sie die Hautprobe auf einem Objektträger und legen Sie dann einen kreisförmigen Deckglas über die Haut ausgeschnitten Probe Anker helfen und glätten das Gewebe für die anschließende digitale Bildbearbeitung. </li><li> Mit den Hautproben von Schritt 4.3, erhalten digitale Bilder von den Haarzellen in jedem Neuromasten der Mitte der Körper Stiche. Nehmen Sie Bilder mit einer Vergrößerung von mindestens 60X und dann zählen die Haare cells innerhalb einzelner Neuromasten für vergleichende quantitative Analyse der Kontroll-und Versuchsgruppen (siehe Abbildung 4). </li></ol>

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This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

Basierend auf den umfangreichen Bestand an Literatur, die für die Analyse der Seitenlinie (LL) Regeneration in embryonalen und larvalen Zebrafisch 8,24,25 festgestellt wurde, das Ziel unserer Studie war es, einen quantitativen Test zur Seitenlinie Regeneration im Zebrafisch zu entwickeln, könnte Krankheitsmodelle, die am besten in der erwachsenen Fische untersucht werden angewendet werden. Wir haben festgestellt, dass bestimmte kritische Punkte sind wichtig, wenn Anwendung von Verfahren für die embryonale ...

Die Seitenlinie (LL)-System ist ein mechanosensorischen Orgel in beiden Fische und Amphibien, die für Gehör, Gleichgewicht, rheotaxis und vermittelnde Verhaltensweisen wie Schul-und Raubvermeidung 5.1 verantwortlich ist gefunden. Es wird der Cluster von Haarzellen von Stützzellen umgeben sind, sind die beide in Strukturen genannt Neuromasten 6 angeordnet. Diese Neuromasten sind in der Regel in vertikalen Linien entlang der Längsachse des Körpers und der Schwanz mit einigen im Kopf der Fische beobachtet horizontale Stiche organisiert (genannt Stiche). Beim Erwachsenen sind Neuromasten in der Anzahl deutlich größer innerhalb der Stiche im Vergleich zu embryonalen oder Fischlarven 6. Biomedizinische Studien haben in Zebrafisch über die Wirkung der Behandlung mit Antibiotika, lärmbedingtem Trauma, chronische Infektion, usw. konzentriert. Haarzellen auf 7,8 in einem Versuch, besser ihre Wirkung beim Menschen zu verstehen. Anders als die meisten Wirbeltiere, teleosts wie Zebrafisch (Danio rerio), haben die Fähigkeit, verlorene Haarzellen zu regenerieren. Zebrafische sind wegen ihrer schnellen Entwicklungszeit und hohe Regenerationsfähigkeit besonders geeignet. Bis heute ist jedoch; Zebrafisch-Studien an der Seitenlinie Entwicklung und / oder der Regeneration haben vor allem die Embryonal-und Larvenstadium Fisch aufgrund der reduzierten Anzahl der Seitenlinie Neuromasten, die für einfachere Zählung und Analyse ermöglicht 6,9,10 genutzt. Allerdings sind viele Zebrafisch-Modelle von neurologischen und nicht-neurologischen Erkrankungen 11-16 in der erwachsenen Fische und nicht die Larven untersucht, konzentrierten wir uns auf die Entwicklung einer Seitenlinie regenerative Test bei erwachsenen Zebrafisch mit Gentamicin (ein Aminoglykosid zuvor in Zebrafisch-Larven und neuerdings mit 17 erwachsenen Fischen verwendet wird), so dass ein Test zur Verfügung, dass den aktuellen erwachsenen Zebrafisch-Krankheitsmodelle angewandt werden könnten. Während früher durch Van Trump e veröffentlichten Verfahrent al. 17. wurden die Bedingungen für die Haarzell Ablation in der erwachsenen Fische, haben sie nicht eine Standardkurve für Neuromasten, die Regeneration für die quantitative Vergleich zwischen Kontroll-und Versuchsgruppen wie bei der Verwendung von transgenen Zebrafischlinien oder pharmakologisch induzierten Krankheitszuständen erforderlich ist, zu etablieren 18 im Zebrafisch. Wir haben daher die Verfahren, gefolgt von Van Trump et al. Für 17 Haarzelle Ablation, aber auf ihre Arbeit gebaut, um eine Standardkurve von Neuromasten Regeneration Ermittlern ermöglichen, unsere Daten, wie mit erwachsenen Zebrafisch Krankheitsmodelle beim Vergleich Kontroll-und Versuchsgruppen einsetzen . Der Assay wurde auch entworfen, um Erweiterung der Analyse auf die einzelnen Haarzelle zu ermöglichen, wenn eine höhere Auflösung erforderlich ist.

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

Aufgrund der klinischen Bedeutung von Hör-und Gleichgewichtsstörungen bei Menschen, Modellorganismen wie Zebrafisch wurden zur Seitenlinie Entwicklung und Regeneration zu studieren. Der Zebrafisch ist wegen seiner schnellen Entwicklungszeit und die hohe Regenerationsfähigkeit für solche Untersuchungen besonders attraktiv. Bisher wurden Zebrafisch-Studien Seitenlinie Regeneration vor allem wegen der geringeren Anzahl von Neuromasten in diesen Phasen genutzt Fische der Embryonal-und Larvenstadien. Dies hat quantitative Analyse der Seitenlinie Regeneration und / oder der Entwicklung leichter in den früheren Entwicklungsstadien gemacht. Da viele Zebrafisch Modelle von neurologischen und nicht-neurologischen Krankheiten im erwachsenen Fisch und nicht im Embryo / Larven untersucht, auf die Entwicklung eines quantitativen Seitenlinie regenerative Assay in erwachsenen Zebrafisch konzentrierten wir uns damit wurde ein Assay zur Verfügung, um Strom angelegt werden kann erwachsenen Zebrafisch Krankheitsmodelle. Aufbauend auf früheren Studien von Van TrumS. et al 17, dass die Verfahren zur Ablation von Haarzellen im erwachsenen mexikanischen blinde Höhlenfische und Zebrafisch (Danio rerio) beschrieben. wurde unser Test zur quantitativen Vergleich zwischen Kontroll-und Versuchsgruppen zu ermöglichen. Dies wurde durch die Entwicklung eines regenerativen Neuromasten Standardkurve, basierend auf dem Prozentsatz der Neuromasten Wieder über einen 24 Stunden Zeitraum nach Gentamicin-induzierte Nekrose von Haarzellen in einem definierten Bereich der Seitenlinie erreicht. Der Assay wurde auch entworfen, um Erweiterung der Analyse auf die einzelnen Haarzellebene, wenn eine höhere Auflösung erforderlich ist, zu ermöglichen.

Optimierung der Verfahren zur Quantifizierung von Neuromasten Regeneration der Seitenlinie bei erwachsenen Zebrafisch. Die Neuromasten von Zebrafisch-Larven sind leicht quantifizierbar; jedoch hat die Seitenlinie des erwachsenen Zebrafisch eine viel größere Anzahl von Neuromasten pro Stich Herstellung quantitative Analysen schwieriger 6,17,19,20. Wie in 1A zu sehen ist, hat der Kopf eine deutlich höhere Anzahl von Neuromasten im Vergleich zu en...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

Da viele Zebrafisch-Modellen der neurologischen und nicht-neurologischen Erkrankungen sind in der erwachsenen Fischen nicht der Embryo / Larven untersucht, entwickelten wir eine quantitative Seitenlinie regenerative Assay, die erwachsenen Zebrafisch Krankheitsmodelle angewendet werden können. Der Test beteiligt Beschluss der 1) Neuromasten und 2) einzelne Haarzelle Ebenen.

Ein Test für Laterallinie Regeneration im erwachsenen Zebrafisch

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K Aufrufe.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  Da viele Zebrafisch-Modellen der neurologischen und nicht-neurologischen Erkrankungen sind in der erwachsenen Fischen nicht der Embryo / Larven untersucht, entwickelten wir eine quantitative Seitenlinie regenerative Assay, die erwachsenen Zebrafisch Krankheitsmodelle angewendet werden können. Der Test beteiligt Beschluss der 1) Neuromasten und 2) einzelne Haarzelle Ebenen. 

Serie: An Assay for Lateral Line Regeneration in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Die Transplantation von Whole Kidney Marrow in Adult Zebrafisch

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Forschung

Biologie

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Zelluläre Toxizität von Nanogenomedicine in MCF-7 Zelllinie: MTT-Assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-Orbital-Injection in Adult Zebrafisch

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

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 ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

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 ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

In Vivo Surface Elektrokardiographie für erwachsene Zebrafische

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.