Questo lavoro è supportato da Howard Hughes Medical Institute e una borsa di studio del NIH per lo studio della ematopoiesi embrionale.

Parte 1. Preparazione del materiale di iniezione <ol><li> Tg (globina: GFP) celle: </li><li> Bleed pesce adulto donatore utilizzando punta della pipetta 10ul rivestito in eparina (1unit/ul) a pescare puntura dietro branchia. </li><li> Aspirare globuli rossi e dispensare nel buffer di cella (0.9x PBS + 5% FBS + 1% Pen / Strep). </li><li> Cellula sospensione filtro su mesh 40uM e determinare la concentrazione di cellule utilizzando emocitometro come precedentemente descritto da LeBlanc et al. </li><li> Spin down e risospendere in buffer cella a concentrazione desiderata in modo tale che il volume di iniezione finale non è più di 5UL. Qui iniettare 1,5-2000000 cellule / destinatario. </li><li> Per le, destrano Texas Red ®, sciogliere il colorante in DPBS per una concentrazione di iniezione finale di 10-12 mg / mL. Piano per iniettare 4 uL per pesce. </li></ol> Parte 2. Iniezione <ol><li> Anestetizzare pesce in tricaine (4.2ml (4mg/ml) tricaine/100ml pesci d&#39;acqua). </li><li> Lavare Hamilton siringa 3-4 volte prima dell&#39;iniezione utilizzando etanolo al 70%. Lavare 3-4 volte con DPBS 0.9x. </li><li> Luogo pesce lato dorsale e rivolta a destra sulla spugna umida. </li><li> Tenere la siringa Hamilton con la mano destra e con il dito indice sullo stantuffo. Delicatamente stabilizzare il corpo del pesce con la mano sinistra. </li><li> La posizione della lancetta con lo smusso rivolto verso l&#39;alto in modo tale che se l&#39;occhio del pesce s fosse un orologio, l&#39;ago sarebbe indicando la posizione 7:00 e con un angolo di 45 gradi per il pesce (figura 1). </li><li> Inserire delicatamente l&#39;ago 1-2 mm in posizione 07:00 e lentamente spingere il pistone. </li><li> Permettono di recuperare il pesce in acqua dolce E3. </li><li> Lavare ago, come descritto sopra, tra le iniezioni di reagenti diversi. </li><li> Flick soluzione cellula per risospendere le cellule ogni pochi minuti per evitare grumi di cellule. </li><li> Mantenere il flusso di pesce per 1 settimana con cambi d&#39;acqua ogni giorno per evitare l&#39;infezione. Noi mantenere i pesci in acqua terapia intensiva per questo periodo (cappotto stress da 10 ml + 5 ml + pemafix melafix 5mL per 38 litri di acqua E3). </li></ol> Parte 3. Rappresentante Risultati Quando eseguito correttamente, è possibile visualizzare materiale iniettato se è stato etichettato in qualche modo. Per esempio, Tg (globina: GFP) globuli rossi dovrebbe essere visto sotto un microscopio a fluorescenza dissezione circola nei vasi dei pesci destinatario casper subito dopo l&#39;iniezione, come illustrato in figura 2. Allo stesso modo, l&#39;iniezione di un destrano 70kDa, coniugato con Texas Red ® possono essere visualizzati nel sistema vascolare del pesce trasparente immediatamente successivo retro-orbitale di iniezione (figura 3). Cellule renali fluorescenti da Tg (β-actina: GFP) pesci donatore può anche essere iniettato retro-orbitalmente in pesci irradiati destinatario casper. Queste cellule finalmente a casa per il midollo destinatario (figura 4a) e può quindi andare a ripopolare il rene dopo diverse settimane (figura 4b). Se l&#39;iniezione è troppo bassa o l&#39;angolo dell&#39;ago bypassa il retro-orbitale cavità dei seni venosi, materiale iniettato può essere visualizzata pooling intorno all&#39;occhio o che scorre fuori di esso. In alternativa, se l&#39;iniezione è troppo profondo, pesce possibile che si verifichino danni ai tessuti o sanguinamento eccessivo, anche se può ancora recuperare. Quando eseguito correttamente, la mortalità dopo retro-orbitale di iniezione deve essere inferiore al 5% del totale pesce iniettato e il successo di consegna alla circolazione sanguigna dovrebbe essere superiore al 90% del totale pesce iniettato. <img src="/files/ftp_upload/1645/1645fig1.jpg" alt="figura 1" /> Figura 1: Illustrazione di retro-orbitale tecnica di iniezione. L&#39;occhio destro del pesce è rappresentato come un orologio analogico in cui l&#39;orologio sette o posizione corrisponde al punto d&#39;iniezione corretta. <img src="/files/ftp_upload/1645/1645fig2.jpg" alt="figura 2" /> Figura 2: Tg (globina: GFP) globuli rossi circolanti nei vasi della coda di un pesce adulto casper tre giorni dopo la retro-orbitale iniezione. <img src="/files/ftp_upload/1645/1645fig3.jpg" alt="figura 3" /> Figura 3: iniezione di successo di una 70kDa destrano coniugato con Texas Red ® usato come colorante fluorescente produce vascolarizzazione nei pesci adulti trasparente, Casper ed è facilmente visualizzati utilizzando standard di tutta la microscopia a fluorescenza animale. <img src="/files/ftp_upload/1645/1645fig4a.jpg" alt="figura 4a" /> Figura 4a: Tre giorni successivi retro-orbitale iniezione di cellule intere midollo rene da Tg (β-actina: GFP) pesci donatore, le cellule possono essere visualizzati homing al midollo reni del combustibile irraggiato pesce destinatario casper adulti. <img src="/files/ftp_upload/1645/1645fig4b.jpg" alt="figura 4b" /> Figura4b: Quattro settimane dopo la retro-orbitale iniezione di cellule intere midollo rene da Tg (β-actina: GFP) pesci donatore, il ripopolamento del rene destinatario con verde cellule renali di tutto midollo del donatore possono essere visualizzati in irradiati pesce destinatario casper adulti.

<ol>
	<li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+route+of+intravenous+drug+delivery+in+a+transgenic+mouse+model.">Comparison of the lateral tail vein and the retro-orbital venous sinus as route of intravenous drug delivery in a transgenic mouse model.</a> Lab Animal. 37, 26-32 (2008).</li><li>Pinkerton, W., Webber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+injecting+small+laboratory+animals+by+the+ophthalmic+plexus+route.">A method of injecting small laboratory animals by the ophthalmic plexus route.</a> Proc. Soc. Exp. Bio. Med. 116, 959-961 (1964).</li><li>White, R. M., Sessa, A. S., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns, C. E., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>LeBlanc, J., Bowman, T. V., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+whole+kidney+marrow+in+adult+zebrafish.">Transplantation of whole kidney marrow in adult zebrafish.</a> J. Vis. Exp. , (2007).</li></ol>

Retro-orbitale iniezione in zebrafish adulto offre la massima efficienza della consegna del materiale iniettato alla circolazione sanguigna con la più bassa incidenza di mortalità. A causa della natura del punto di iniezione, la puntura sembra guarire in fretta, diminuendo l&#39;insorgenza di infezioni e la quantità di perdita di sangue. Il sito può anche essere iniettato ripetutamente nel corso di un breve periodo di tempo per iniezioni giornaliere di farmaci, per esempio. Grandi quanti...

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<td>Heparin sodium salt from porcine intestinal mucosa</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>2106</td>
<td>Preweighed vial of 300 USP units</td>
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<td>Tricaine-S</td>
<td>&nbsp;</td>
<td>Western Chemical</td>
<td>&nbsp;</td>
<td>MS-222</td>
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<td>Dulbecco&#x2019;s Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-144</td>
<td>&nbsp;</td>
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<td>Dextran, Texas Red&reg;</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
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<td>E3</td>
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<td>&nbsp;</td>
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0.17 mM Kcl 
0.33 mM CaCl2 
0.33 mM MgSO2
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<td>Microliter Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
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retro-orbital injection in adult zebrafish

Trattamento farmacologico degli animali tutto è uno strumento essenziale in qualsiasi sistema modello per studi di genetica e chimica farmacologica. Per via endovenosa (ev) è spesso la forma più efficace e non invasiva di consegna di un agente di interesse. Nel pesce zebra (<em&gt; Danio rerio</em&gt;), Iniezione IV di farmaci è stata a lungo una sfida a causa del diametro del vaso di piccole dimensioni. Questo si è dimostrato un ostacolo significativo per l&#39;iniezione di cellule durante il trapianto di cellule staminali emopoietiche. Storicamente, le iniezioni nel sangue sono state fatte direttamente al cuore. Tuttavia, questa intra-cardiaca procedura ha un tasso di mortalità molto alto, come il cuore è spesso perforato durante l&#39;iniezione di lasciare il pesce a rischio di infezione, perdita massiccia di sangue o di danno d&#39;organo fatale. Basandoci sulla nostra esperienza con il mouse, abbiamo sviluppato una nuova procedura di iniezione nel pesce zebra in cui il sito di iniezione è dietro l&#39;occhio e nel retro-orbitale del seno venoso. Questo retro-orbitale (RO), tecnica di iniezione è stato impiegato con successo in entrambi l&#39;iniezione di farmaci nei pesci adulti, nonché il trapianto di cellule del midollo intero rene. Iniezione RO ha un tasso di mortalità molto più basso rispetto ai tradizionali iniezione intra-cardiaca. I pesci che vengono iniettati retro-orbitalmente tendono a sanguinare iniezione meno seguito e sono ad un rischio molto più basso di lesioni per un organo importante come il cuore. Altre cellule, se eseguito correttamente, iniettati e / o farmaci entrare direttamente nel flusso sanguigno che permette composti di esercitare il loro effetto sul pesce intero e le cellule renali con facilità a casa per la loro nicchia. Così, questa nuova tecnica di iniezione riduce la mortalità, consentendo una distribuzione efficiente del materiale nel sangue dei pesci adulti. Qui esemplificare questa tecnica retro-orbitale iniezione di Tg (<em&gt; Globina</em&gt;: GFP) in cellule adulte<em&gt; Casper</em&gt; Pesci così come l&#39;iniezione di un colorante rosso fluorescente (destrano, Texas Red<em&gt;</em&gt;) In adulti<em&gt; Casper</em&gt; Pesce. Abbiamo poi visualizzare le iniezioni di successo al microscopio a fluorescenza intero animale.

Watch this Scientific Journal Video about Retro-orbital Injection in Adult Zebrafish at JoVE.com

Retro-orbital Injection in Adult Zebrafish

Qui vi mostriamo come fare retro-orbitale iniezione in zebrafish adulti.

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 ...

retro-orbital-injection-in-adult-zebrafish

Research

JoVE Journal

Biology

24.6K Views.  Children’s Hospital Boston. Qui vi mostriamo come fare retro-orbitale iniezione in zebrafish adulti.

Video: Retro-orbital Injection 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

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Cervello iniezione ventricolo

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissezione di organi dal Zebrafish adulti

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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, ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissezione del rene Zebrafish adulti

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...