The authors have nothing to disclose.

Tutte le procedure vengono eseguite seguendo le linee guida descritte in &quot;Principi di cura degli animali da laboratorio&quot; (National Institutes of pubblicazione della Sanità n. 85-23, riveduta 1985) e il protocollo di animali Rosalind Franklin dell&#39;Università Institutional Animal Care ed uso commissione ha approvato 08-19. 1. Gentamicina-induzione di capelli necrosi cellulare <ol><li> Preparare gentamicina solfato in soluzione fisiologica ad una concentrazione finale di 0,004% (4,32 mM). </li><li> Mettere pesci adulti (D. rerio, 4-6 mesi di età) in un recipiente contenente il 0,004% (4.32 mM) soluzione gentamicina. Ogni contenitore può essere utilizzato, ma si usa un contenitore pesce da un sistema acquatico Pharmacal che è in larga 7, 6 in alto, e 7 a lungo. Mettere il recipiente con il pesce in un incubatore a 28 ° C per 24 ore. Impostare il volume totale del fluido nel serbatoio ad un livello sufficiente a mantenere il pesce in uno Stato idoneo per il periodo di 24 ore. Nota: L&#39;aerazione del fluido gentamicina non è necessario se viene utilizzato un volume sufficiente per il numero di pesci in trattamento. </li></ol> 2. Colorazione vitale delle cellule ciliate <ol><li> Preparare una concentrazione 0,08% (in soluzione fisiologica) del colorante vitale fluorescente [4-4-diethylaminostyryl)-N-metilpiridinio ioduro (485 nm di eccitazione e 603 nm λ λ emissione in metanolo) a partire da una soluzione madre di lavoro di 15 mg / ml in etanolo. </li><li> Per determinare se il trattamento è stato efficace gentamicina un sottoinsieme di controllo e gentamicina pesci trattati sono macchiati immediatamente posizionando pesce nel pozzetto di una piastra di coltura 6 pozzetti contenenti il ​​colorante vitale. Utilizzare un numero sufficiente di pesci (e piastre di coltura come richiesto per la significatività statistica da raggiungere. Basato sulla velocità dell&#39;esaminatore del conteggio neuromast, mettere il pesce nei piatti in modo sfalsato nel tempo in modo che i pesci non sono macchiati per oltre 75 min come descritto al punto 2.3. </li><li> Posizionare le piastre dal punto 2.2 in un cassetto panchina dal microscopio a fluorescenza peressere utilizzato per l&#39;esame di neuromasti macchiati. Spegnere le luci della stanza per evitare tempra del colorante vitale nel periodo colorazione 1 ora a temperatura ambiente. </li><li> Preparare sia dye wash-out e serbatoi d&#39;acqua anestetici. Dye acqua wash-out è l&#39;acqua del pesce normale e per l&#39;acqua anestetico, aggiungere sufficienti 2-fenossietanolo in modo che una diluizione 1:1000 in acqua il pesce normale è raggiunto. </li><li> Disporre il pesce in acqua i pesci normali eccesso di sciacquare eccesso di colorante vitale e passare al punto 3.1 per osservazione di colorante vitale macchiato di pesce. </li><li> Per esaminare la rigenerazione di neuromasti, trasferire pesce gentamicina trattati che sono stati lavati in acqua pesce normale tra un incubatore per 8-16 ore a 28 ° C. </li><li> In tempi diversi tra 8-16 h, pesce vengono rimossi dal termostato, lavate e colorate come indicato nei passaggi 2.1-2.4. Passare al punto 3.1 per osservazione di colorante vitale macchiato di pesce. </li></ol> 3. Anestetizzante Fish and Counting fluorescente di neuromasti <ol><li&gt; Asciugare ogni pesce su un tovagliolo di carta per rimuovere il liquido in eccesso e poi metterlo su un foglio di carta inumidito filtro che è centrata sul coperchio di una piastra di Petri di plastica. </li><li> Mettere il coperchio sul palco di uno stereo microscopio a fluorescenza per ottenere un&#39;immagine digitale del colorante vitale neuromasti macchiati dei punti del corpo metà. </li><li> Utilizzare una fotocamera digitale posto sulla stereo microscopio a fluorescenza impostato un ingrandimento di 2X per acquisire immagini per la successiva analisi quantitativa. Nota: L&#39;impostazione di ingrandimento del microscopio stereo può dipendere dalla marca di microscopio utilizzato, ma l&#39;impostazione dovrebbe consentire una facile visualizzazione e il conteggio dei singoli neuromasti all&#39;interno della metà dei punti del corpo. </li><li> Determinare la quantità di rigenerazione contando il numero di neuromasti visibili nei quattro punti designati sul lato più in basso ventrale del pesce appena prossimale verso destra pinna pettorale (vedi Figura 1). Per l&#39;analisi statistica utilizzare un test adeguato, come ANOVA or T-test di Student. Gli esperimenti dovrebbero utilizzare un minimo di 5 pesci al punto di tempo e di tutti gli esperimenti devono essere ripetute un minimo di 3x. </li><li> Sulla base della curva rigenerazione neuromast (vedere Figura 3), contare neuromasti tra 8-16 hr alberino gentamicina wash-out di essere all&#39;interno della fase lineare della curva di rigenerazione. Nota: L&#39;uso della fase di tempo lineare permette per una corretta analisi quantitativa tra il controllo e gruppi sperimentali. </li></ol> 4. Conteggio fluorescente delle cellule ciliate individuale per ottenere una maggiore risoluzione delle analisi quantitativa se l&#39;analisi Neuromast non è statisticamente significativo <ol><li> Se l&#39;analisi quantitativa a livello di neuromasti non è significativo, analisi a livello della cellula capelli individuo può essere utilizzato anche per ottenere un grado di risoluzione più elevata. Selezionare il pesce in un punto particolare tempo di post gentamicina wash-out (punto nel tempo in base agli studi neuromast precedenti), colorante vitale stain il pesce come descritto nel protocollo 2, e quindi eutanasia il pesce con 2-fenossietanolo a una diluizione 1:500 per 1-5 min. </li><li> Nella luce soffusa per evitare tempra, fare quattro incisioni in modo che un preparato lembo cutaneo quadrata è composto come segue. Effettuare un&#39;incisione lungo le costole superiori del pesce fino a quando non è allineato con le pinne anali, quindi fare un&#39;incisione attraverso la pancia, e, infine, fare due incisioni verticali su ciascun lato di queste incisioni così si crea il lembo cutaneo piazza. Nota: Questa preparazione pelle integrare i punti del corpo metà utilizzati negli esperimenti neuromast. </li><li> Porre il campione di pelle su un vetrino e poi posto un coperchio circolare vetro scivolare sul campione di pelle asportati per aiutare ancoraggio e appiattire il tessuto per il successivo immagini digitali. </li><li> Utilizzando i campioni di pelle dal punto 4.3, di ottenere immagini digitali delle cellule ciliate all&#39;interno di ogni neuromast dei punti del corpo metà. Prendete le immagini ad un ingrandimento di almeno 60X e poi contare il ce capellills all&#39;interno dei singoli neuromasti di analisi quantitativa comparativa del controllo e gruppi sperimentali (vedi Figura 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.

Basato sulla vasta letteratura che è stato stabilito per l&#39;analisi della linea laterale (LL) rigenerazione embrionale e larvale zebrafish 8,24,25, l&#39;obiettivo del nostro studio è stato quello di sviluppare un test quantitativo per la linea rigenerazione laterale in zebrafish che potrebbe essere applicato a modelli di malattia che sono meglio studiati nei pesci adulti. Abbiamo scoperto che alcuni punti critici sono importanti quando si applicano le procedure sviluppate per embrionale pesce / larvale ...

Il sistema (LL) linea laterale è un organo mechanosensory trovato in entrambi i pesci e anfibi che è responsabile per l&#39;udito, l&#39;equilibrio, rheotaxis e comportamenti di mediazione, come la scuola e il predatore evasione 1-5. Si compone di gruppi di cellule capelli circondate da cellule di supporto, entrambi i quali sono disposti in strutture chiamate neuromasti 6. Questi neuromasti sono tipicamente organizzati in linee verticali (chiamato punti) lungo l&#39;asse longitudinale del corpo e la coda con alcuni punti orizzontali osservati nella testa del pesce. Nell&#39;adulto, neuromasti sono significativamente più numerosi nei punti rispetto ai pesci embrionale o larvale 6. Studi biomedici in zebrafish si sono concentrati sugli effetti del trattamento antibiotico, trauma indotta dal rumore, infezione cronica, ecc. su cellule ciliate 7,8 nel tentativo di capire meglio i loro effetti sugli esseri umani. Diversamente dalla maggior parte dei vertebrati, TEleosts, come il pesce zebra (Danio rerio), hanno la capacità di rigenerare le cellule dei capelli perduti. Zebrafish sono particolarmente utili perché del loro tempo rapido sviluppo e ad alta capacità rigenerativa. Ad oggi, tuttavia, studi sullo sviluppo di zebrafish linea laterale e / o rigenerazione sono principalmente utilizzati il pesce stadio embrionale e larvale causa del numero ridotto di neuromasti linea laterale che agevola conteggio e analisi 6,9,10. Tuttavia, come molti modelli di zebrafish di malattie neurologiche e non neurologiche 11-16 sono studiate nei pesci adulti e non le larve, ci siamo concentrati sullo sviluppo di un test rigenerativa linea laterale in zebrafish adulto con gentamicina (un aminoglicoside precedentemente utilizzato in larve di zebrafish e più recentemente utilizzato con pesci adulti 17) in modo che un saggio era disponibile che potrebbe essere applicato a modelli di malattia di zebrafish adulto attuali. Mentre in precedenza pubblicati procedure di Van Trump et al. 17 ha stabilito le condizioni per l&#39;ablazione delle cellule dei capelli nel pesce adulto, che non hanno stabilito una curva standard per la rigenerazione neuromast che è necessario per il confronto quantitativo tra controllo e gruppi sperimentali come quando si utilizzano linee di zebrafish transgenici o stati di malattia farmacologicamente indotti in zebrafish 18. Abbiamo quindi seguito le procedure di Van Trump et al. 17 per l&#39;ablazione delle cellule dei capelli, ma costruito sul loro lavoro per stabilire una curva standard di rigenerazione neuromast per consentire ai ricercatori di utilizzare i nostri dati quando si confrontano controllo e gruppi sperimentali come ad esempio con modelli di malattia di zebrafish adulto . Il dosaggio è stato inoltre progettato per consentire estensione dell&#39;analisi alla cella capelli individuo quando è richiesto un livello di risoluzione.

<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

Data l&#39;importanza clinica dell&#39;udito e disturbi dell&#39;equilibrio nell&#39;uomo, organismi modello come zebrafish sono stati utilizzati per studiare lo sviluppo linea laterale e rigenerazione. Il zebrafish è particolarmente attraente per tali studi a causa del suo tempo di sviluppo rapido e la sua elevata capacità rigenerativa. Ad oggi, gli studi di zebrafish della linea di rigenerazione laterali sono principalmente utilizzati pesci delle fasi embrionali e larvali a causa del numero inferiore di neuromasti in queste fasi. Ciò ha reso analisi quantitativa della linea rigenerazione e / o sviluppo più semplice negli stadi precoci di sviluppo laterale. Poiché molti modelli di zebrafish di malattie neurologiche e non neurologiche sono studiate nei pesci adulti e non nell&#39;embrione / larve, ci siamo concentrati sullo sviluppo di un laterale dosaggio linea rigenerativa quantitativa in zebrafish adulto in modo che un test era disponibile che potrebbe essere applicato a corrente modelli di malattia di zebrafish adulto. Basandosi su studi precedenti di Van Trump et al. 17 che descrive le procedure per l&#39;ablazione delle cellule ciliate adulto messicano pesci ciechi grotta e zebrafish (Danio rerio), il nostro test è stato progettato per permettere il confronto quantitativo fra controllo e gruppi sperimentali. Ciò è stato realizzato attraverso lo sviluppo di una curva standard neuromast rigenerativa basata sulla percentuale di ricomparsa neuromast su un periodo di tempo di 24 ore seguente gentamicina indotta necrosi delle cellule ciliate in una regione definita della linea laterale. Il dosaggio è stato inoltre progettato per consentire estensione dell&#39;analisi a livello di cella capelli individuo quando è richiesto un livello di risoluzione.

Ottimizzazione delle procedure per quantificare la rigenerazione neuromast della linea laterale in zebrafish adulto. Le neuromasti di zebrafish larvale sono facilmente quantificabili; Tuttavia, la linea laterale del zebrafish adulto ha un numero molto maggiore di neuromasti per punto effettuare analisi quantitative più difficile 6,17,19,20. Come si vede nella Figura 1A, la testa ha un numero significativamente maggiore di neuromasti rispetto sia ...

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

Poiché molti modelli di zebrafish di malattie neurologiche e non neurologiche sono studiate nei pesci adulti piuttosto che l&#39;embrione / larve, abbiamo sviluppato una linea laterale rigenerativo dosaggio quantitativo che può essere applicato a modelli di malattia di zebrafish adulto. Il saggio coinvolto risoluzione al neuromast 1) e 2) livelli di cellule singolo capello.

Un test per Lateral Rigenerazione Line in Zebrafish adulti

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 Views.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  Poiché molti modelli di zebrafish di malattie neurologiche e non neurologiche sono studiate nei pesci adulti piuttosto che l'embrione / larve, abbiamo sviluppato una linea laterale rigenerativo dosaggio quantitativo che può essere applicato a modelli di malattia di zebrafish adulto. Il saggio coinvolto risoluzione al neuromast 1) e 2) livelli di cellule singolo capello. 

Video: 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.

Il trapianto di midollo renale intero in Zebrafish adulti

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

Ricerca

Biologia

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.

Tossicità cellulare di Nanogenomedicine in MCF-7 Linea Cell: saggio MTT

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-orbitale iniezione in Zebrafish adulti

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

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Iniezione intraperitoneale in Zebrafish adulti

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

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 elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

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 elettroporazione in vivo di Morpholinos nella Adult Regenerating 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.

Il prelievo di sangue per l&#39;analisi biochimica in Zebrafish Adult

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 Zebrafish adulti

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.

Ripetuto il prelievo di sangue per esami del sangue in Zebrafish adulti

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.

Elettrocarcardio in superficie in vivo per il pesce zebra adulto

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