The authors have nothing to disclose.

Todos os procedimentos são realizados seguindo as diretrizes descritas em &quot;Princípios de Laboratório Animal Care&quot; (Institutos Nacionais de Saúde não publicação. 85-23, revisado em 1985) e do protocolo de animais Comitê Institucional Animal Care e Use Rosalind Franklin University aprovado 08-19. 1. Gentamicina-indução de necrose das células do cabelo <ol><li> Prepare de sulfato de gentamicina em solução salina normal com uma concentração final de 0,004% (4,32 mM). </li><li> Coloque o peixe adulto (D. rerio, 4-6 meses de idade) em um recipiente que contém a 0,004% (4,32 mM) solução de gentamicina. Qualquer recipiente pode ser usado, mas utiliza um recipiente de peixe a partir de um sistema aquático Pharmacal que é 7, em largura, 6 de alta, e em 7 de comprimento. Colocar o recipiente com o peixe numa incubadora a 28 ° C durante 24 hr. Definir o volume total de fluido no tanque a um nível suficiente para manter os peixes num estado viável para o período de 24 horas. Nota: O arejamento do fluido de gentamicina não é necessary se volume suficiente é usado para o número de peixes a serem tratados. </li></ol> 2. Coloração Vital das células ciliadas <ol><li> Prepara-se uma concentração de 0,08% (em solução salina normal) do corante vital fluorescente [4-4-diethylaminostyryl iodeto)-N-metilpiridínio (485 nm de excitação e 603 nm λ λ emissão em metanol) a partir de uma solução de estoque de trabalho de 15 mg / ml em etanol. </li><li> Para determinar se o tratamento foi eficaz gentamicina um subconjunto de controlo e gentamicina peixes tratados são corados imediatamente através da colocação de peixe no poço de uma placa de 6 poços de cultura que contém o corante vital. Use um número suficiente de peixe (e placas de cultura, conforme exigido para significância estatística a ser alcançado. Baseado na velocidade do examinador de contagem neuromasto, coloque o peixe nas placas de forma escalonada ao longo do tempo para que os peixes não estão manchadas por mais de 75 min tal como descrito na etapa 2.3. </li><li> Coloque as placas a partir do passo 2.2 em uma gaveta banco pelo microscópio de fluorescência paraser utilizados para exame de neuromastos coradas. Desligue as luzes da sala para evitar a extinção do corante vital durante o período de coloração 1 hora em temperatura ambiente. </li><li> Prepare tanto corante wash-out e tanques de água anestésicos. Dye lavar-out água é água peixe normal e para água anestésico, adicione suficiente 2-fenoxietanol para que uma diluição 1:1.000 em peixes de água normal é alcançado. </li><li> Coloque o peixe na água em excesso peixes normais para enxaguar corante vital excesso e avançar para o passo 3.1 para observação de corante vital manchado peixes. </li><li> Para examinar a regeneração de neuromastos, transferir tratados pela gentamicina peixe que foram lavados em água de peixe normal de uma incubadora para entre 8-16 horas a 28 ° C. </li><li> Em diversos momentos entre 8-16 horas, os peixes são removidos da incubadora, lavado e corados tal como indicado nos passos 2,1-2,4. Avance para o passo 3.1 para observação de corante vital manchado peixes. </li></ol> 3. Anestesiar Fish and Counting fluorescente de neuromastos <ol><li&gt; Seque cada peixe em um papel toalha para remover o excesso de líquido e, em seguida, colocá-lo em um pedaço de papel de filtro umedecido que é centrada na tampa de uma placa de Petri de plástico. </li><li> Colocar a tampa sobre a plataforma de um microscópio estéreo fluorescente para obter uma imagem digital das corante neuromastos coradas vitais dos pontos do corpo médio. </li><li> Use uma câmera digital colocada no microscópio estéreo fluorescente definir uma ampliação de 2X para capturar imagens para análise quantitativa posterior. Nota: A configuração de ampliação do microscópio estéreo pode depender da marca do microscópio usado, mas a definição deve permitir uma fácil visualização e contagem de neuromastos individuais dentro de meados dos pontos do corpo. </li><li> Determinar a quantidade de regeneração através da contagem do número de neuromastos visíveis dentro dos quatro pontos designados no lado inferior mais ventral do peixe apenas proximal à barbatana peitoral direita (ver figura 1). Para a análise estatística utilizar um ensaio apropriado, tal como o ANOVAr T-teste de Student. Experimentos deve utilizar um mínimo de 5 peixes por ponto de tempo e todos os experimentos devem ser repetidos um mínimo de 3x. </li><li> Com base na curva de tempo de regeneração neuromasto (ver Figura 3), a contagem de neuromastos entre 8-16 horas pós gentamicina lava--se na fase linear da curva de regeneração. Nota: O uso da fase de tempo linear permite a análise quantitativa adequada entre os grupos controle e experimentais. </li></ol> 4. Counting fluorescentes das células ciliadas individual para obtenção de maior resolução da Análise Quantitativa Se a análise neuromasto não é estatisticamente significativa <ol><li> Se a análise quantitativa no nível de neuromastos não é significativa, a análise ao nível da célula individual do cabelo também pode ser utilizada para obter um elevado grau de resolução. Selecione peixe num determinado ponto de tempo pós gentamicina wash-out (ponto de tempo com base nos estudos anteriores neuromasto), corante vital ruaain o peixe, conforme descrito no Protocolo 2, e depois sacrificá o peixe usando 2-fenoxietanol na diluição de 1:500 por 1-5 min. </li><li> Na luz suave para evitar extinção, fazer quatro incisões para que uma preparação retalho de pele quadrado é feita da seguinte forma. Faça uma incisão ao longo das costelas superiores do peixe até que esteja alinhado com as barbatanas anais, em seguida, fazer uma incisão em toda a barriga e, finalmente, fazer duas incisões verticais de cada lado dessas incisões para que o retalho de pele quadrado é criado. Nota: Esta preparação da pele irá incorporar os pontos do corpo meados utilizados nos experimentos neuromasto. </li><li> Coloque a amostra de pele numa lâmina de vidro e, em seguida, coloque uma circular lamínula de vidro sobre a amostra de pele excisadas para ajudar a ancorar e achatar o tecido de imagem digital subseqüente. </li><li> Utilizando as amostras de pele a partir do passo 4.3, obter imagens digitais de células ciliadas em cada neuromasto dos pontos do corpo médio. Tire fotos com uma ampliação de pelo menos 60X e depois contar o ce cabelolls dentro neuromastos individuais para análise quantitativa comparativa dos grupos controle e experimental (ver 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.

Com base no extenso corpo de literatura que foi estabelecido para a análise da linha lateral (LL) em regeneração embrionário e larval do peixe-zebra 8,24,25, o objetivo de nosso estudo foi o desenvolvimento de um ensaio quantitativo para a regeneração da linha lateral em peixes-zebra que poderia ser aplicada aos modelos de doenças que são mais bem estudados no peixe adulto. Descobrimos que certos pontos críticos são importantes quando se aplica procedimentos desenvolvidos para peixes embrionário / ...

A linha lateral sistema (LL) é um órgão mecanosensorial encontrado em ambos os peixes e anfíbios que é responsável pela audição, equilíbrio, reotaxia e comportamentos de mediação, tais como escolaridade e evitar predadores 1-5. É composta de agregados de células ciliadas rodeadas por células de suporte, os quais estão posicionados em estruturas chamadas neuromastos 6. Estes neuromastos são normalmente organizados em linhas verticais (chamados pontos) ao longo do eixo longitudinal do corpo e cauda com alguns pontos horizontais observados na cabeça do peixe. No adulto, neuromastos são significativamente maiores em número dentro dos pontos em comparação com as de peixes embrionários ou larvar 6. Estudos biomédicos no peixe-zebra têm-se centrado sobre o efeito do tratamento com antibióticos, trauma induzida por ruído, infecção crônica, etc. em células ciliadas 7,8, numa tentativa de compreender melhor os seus efeitos nos seres humanos. Diferentemente da maioria dos vertebrados, teleosts, tais como peixe-zebra (Danio rerio), têm a capacidade de regenerar células capilares perdidas. Peixe-zebra são particularmente úteis por causa do seu tempo de desenvolvimento rápido e elevada capacidade regeneradora. Até o momento, no entanto; estudos sobre o desenvolvimento de peixe zebra linha lateral e / ou regeneração têm utilizado principalmente o peixe estágio embrionário e larval, devido ao número reduzido de neuromastos linha lateral que facilita a contagem e análise 6,9,10. No entanto, como muitos modelos de peixe-zebra de doenças neurológicas e não neurológicas 11-16 são estudadas no peixes adultos e as larvas não, nós nos concentramos no desenvolvimento de um ensaio de regeneração da linha lateral no peixe-zebra adulto usando gentamicina (um aminoglicosídeo usado anteriormente em larvas do peixe e mais recentemente usado com peixe adulto 17), de modo que um ensaio estava disponível que poderia ser aplicada para adultos modelos actuais de doenças de peixes-zebra. Embora os procedimentos publicados anteriormente por Van Trump et al. 17 estabeleceu as condições para a ablação de células ciliadas no peixe adulto, eles não estabelecer uma curva padrão para a regeneração neuromasto que é necessário para comparação quantitativa entre os grupos controle e experimentais, como a utilização de linhas de zebrafish transgênicos ou estados patológicos induzidos farmacologicamente em peixes-zebra 18. Por isso, seguiu os procedimentos de Van Trump et al. 17 para ablação de células ciliadas, mas construída sobre o seu trabalho para estabelecer uma curva padrão de regeneração neuromasto para permitir aos investigadores usar os nossos dados ao comparar os grupos controle e experimentais, tais como modelos de doenças com peixe-zebra adulto . O ensaio também foi concebida para permitir a extensão da análise para a célula de cabelo individual, quando é necessário um elevado nível de resolução.

<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

Devido à importância clínica da audição e no equilíbrio do homem, de organismos-modelo, como o peixe-zebra foram usados ​​para estudar o desenvolvimento da linha lateral e regeneração. O peixe-zebra é particularmente atraente para tais estudos por causa de seu tempo de desenvolvimento rápido e sua alta capacidade de regeneração. Até o momento, os estudos de peixe-zebra de regeneração da linha lateral têm utilizado principalmente peixes dos estágios embrionários e larvais por causa do menor número de neuromastos nestes estágios. Isso fez com que a análise quantitativa da linha de regeneração e / ou mais fácil o desenvolvimento nas fases de desenvolvimento anteriores lateral. Porque muitos modelos de peixe-zebra de doenças neurológicas e não neurológicas são estudados os peixes adultos e não no embrião / larvas, que focada no desenvolvimento de um ensaio quantitativo linha regenerativa lateral, no peixe-zebra adultos de modo que um ensaio estava disponível que poderia ser aplicada a corrente modelos de doenças zebrafish adultos. Construir em estudos anteriores por Van Trump et al. 17, que descreve os procedimentos de ablação de células ciliadas adulto mexicano peixes cegos da caverna e peixe-zebra (Danio rerio), nosso ensaio foi concebido para permitir a comparação quantitativa entre os grupos controle e experimentais. Isto foi conseguido através do desenvolvimento de uma curva padrão neuromasto regenerativo com base na percentagem de reaparecimento neuromasto ao longo de um período de tempo de 24 horas a seguir a necrose induzida por gentamicina de células ciliadas em uma região definida da linha lateral. O ensaio também foi concebida para permitir a extensão da análise ao nível da célula individual do cabelo quando é necessário um elevado nível de resolução.

Otimização dos procedimentos para quantificar a regeneração neuromasto da linha lateral no peixe-zebra adulto. Os neuromastos de zebrafish larval são facilmente quantificáveis; no entanto, a linha lateral do peixe-zebra adulto tem um número muito maior de neuromastos por ponto fazendo análises quantitativas mais difícil 6,17,19,20. Como visto na Figura 1A, a cabeça tem um número significativamente maior de neuromastos comparação com q...

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

Porque muitos modelos de peixe-zebra de doenças neurológicas e não neurológicas são estudadas no peixe adulto, em vez de o embrião / larvas, foi desenvolvido um ensaio quantitativo regenerativa linha lateral, que pode ser aplicada aos modelos de doenças de peixes-zebra adultos. O ensaio envolveu a resolução neuromasto 1) e 2) os níveis de células de cabelo individuais.

Um ensaio para a lateral de Regeneração de Linha em Zebrafish Adulto

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 visualizações.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  Porque muitos modelos de peixe-zebra de doenças neurológicas e não neurológicas são estudadas no peixe adulto, em vez de o embrião / larvas, foi desenvolvido um ensaio quantitativo regenerativa linha lateral, que pode ser aplicada aos modelos de doenças de peixes-zebra adultos. O ensaio envolveu a resolução neuromasto 1) e 2) os níveis de células de cabelo individuais. 

Vídeo: 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.

Transplante de Medula Renal inteiro em Zebrafish Adulto

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

Biologia

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 de injeção no Zebrafish Adulto

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.

Injeção intraperitoneal em Zebrafish Adulto

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.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

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.

Na eletroporação in vivo de Morpholinos no Adulto Regeneração Zebrafish Cauda 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.

Coleta de sangue para análise bioquímica em Zebrafish Adulto

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 ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualização de Desenvolvimento Craniofacial nas SOX10: Kaede Transgênicos Zebrafish Linha Usando Time-lapse microscopia confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

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 Adulto

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

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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.

Colheita de sangue para testes repetidos de sangue em Zebrafish Adulto

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