This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

Todos os procedimentos com animais foram aprovados pelo Comitê de Ética da Universidade de Mie o, e foram realizados de acordo com a regulamentação bem-estar animal japonês &#39;Ato de Bem-Estar e Gestão de Animais&#39; (Ministério do Meio Ambiente do Japão) e tenha cumprido as normas internacionais. 1. Preparação da agulha NOTA: Todos os experimentos foram realizados sob anestesia, e foram feitos todos os esforços para minimizar o sofrimento. Para a eutanásia, os peixes foram imersos num banho de gelo-água (5 partes de gelo / 1 parte de água em ≤4 ° C) durante ≥20 min. <ol><li> Prepare as agulhas microcapilares vidro puxando um capilar de vidro de 1.0 mm de diâmetro exterior, com um puxador de agulha (Figura 1A). </li><li> Corte as pontas das agulhas obliquamente utilizando uma tesoura fina (Figura 1B). O diâmetro da ponta ideal deve ser de aproximadamente 100 - 200 um (Figura 1C). Se o diamete pontar é demasiado estreito, o sangue não vai entrar na agulha. </li><li> Dissolve-se heparina em solução salina a uma concentração de 5 mg / ml. </li><li> Coloque uma agulha pré-cortados no final nosepiece de um conjunto do tubo de aspirador e mantenha o bocal na boca, ou se conectar a agulha para um dispensador de lâmpada. Mergulhe a ponta da agulha para a solução de heparina, e heparinizar a agulha por sucção e soprando através da solução (Figura 1D e 1E). Nota: O conjunto do tubo de aspirador foi usado para a criopreservação de esperma de peixe-zebra 16. Tubo de borracha longa pode parar qualquer sangue voando na boca. A definição de um filtro no meio do caminho do tubo pode evitar os perigos. </li><li> Armazenar as agulhas heparinizados em um prato de Petri de 10 cm e seco ao ar durante pelo menos 1 h. Um grande número de agulhas pode ser preparado antecipadamente. </li></ol> 2. Anestesia <ol><li> Preparar a solução de anestésico numa pequena caixa de plástico através da mistura de 200 ml de água de peixe com 100 ul de 2-fenoxietanol (2-PE). A concentração final dos anestésicos é de 500 ppm. CUIDADO! 2-PE tem um efeito rápido. </li><li> Remover número desejado de peixe (estirpe AB) a partir do sistema de circulação. </li><li> Use uma rede para transferir o peixe para os anestésicos para 1-2 min (Figura 1F). Observar os peixes nadar gradualmente, espalhar as barbatanas peitorais horizontalmente, suspiro, e tem movimentos rápidos opérculo dentro de 1 min. </li><li> À medida que o tempo passa, observar o peixe estava deitado no fundo do caso e finalmente parar de nadar. O plano cirúrgico de anestesia chegará quando o peixe pára para respirar e os movimentos são lentos opérculo. Neste ponto, o peixe está pronto para a coleta de sangue. </li><li> Usando uma escumadeira levantar o peixe anestesiados a partir do 2-PE e suavemente coloque-o sobre uma toalha de papel embebido com os anestésicos (Figura 1G). Cubra a cabeça do peixe com papel de tecido macio também embebido com solução de 2-PE para prevenir o ressecamento dos olhos e usar outro papel de tecido macio e seco para gentilmentesecar a superfície do corpo. </li></ol> 3. Recolha de Sangue <ol><li> Coloque uma agulha heparinizado no final nosepiece do conjunto do tubo de aspiração (ou o bulbo dispensador) e segure a extremidade do bocal do conjunto do tubo de aspiração na boca. </li><li> Segure a extremidade nosepiece ea agulha juntos, e retire cuidadosamente as escalas que interferem com a ponta da agulha. Inserir a agulha em um 30 - ângulo de 45 ° para o local de coleta de sangue. Evitar a perfuração do tracto gastrointestinal (Figura 1H). Em caso de utilização de bulbo dispenser, pressione a lâmpada com o polegar eo dedo médio, e bloquear o buraco na ponta do bulbo com primeiro dedo, em seguida, inserir a agulha conforme descrito acima. Nota: O local para a recolha de sangue é ao longo do eixo do corpo e do posterior para o ânus na região da aorta dorsal. A aorta dorsal (DA) e a veia cardinal posterior (PCV) são apenas ventral para a coluna vertebral (Figura 2). </li><li> Começar a sugar obocal extremidade do conjunto do tubo de aspiração, quando a agulha é sentida tocar na coluna vertebral. Se o sangue não subir, mova a ponta da agulha sutilmente com a mão para estimular o fluxo sanguíneo. Note que quando o sangue está a aumentar na agulha, pare imediatamente a tremer e sugar delicadamente (Figura 1I). Em caso de utilização de bulbo dispensador, libertar a pressão do bolbo para aspirar o sangue. </li><li> Observar o sangue irá subir lentamente para dentro da agulha de uma maneira pulsátil, sem aspiração, o qual é provável, porque a pressão do sangue arterial. Assim, não é necessário para sugar se a agulha penetra correctamente a artéria. </li><li> Pare sucção depois o volume adequado de sangue é recolhido. Retire a agulha do peixe e pressione o local da punção usando lenço de papel macio para evitar qualquer sangramento. Observe a parada do sangramento após cerca de 10-20 segundos de pressão do dedo (Figura 1J). </li><li> Após o sangramento é interrompido, transferir imediatamente o peixe de volta a um cleum em água quente (~ 28 ° C) do tanque. Ajude o peixe para recuperar suavemente por água de roda para as brânquias até que ele começa a nadar. Nota: Mantenha-se 5 postsampling peixe em um tanque de 2 L e conecte o tanque para o sistema de circulação para oxigenar o peixe. A adição de antibióticos para o peixe de água não é necessária. Manter o peixe com caixa normal e alimentação. </li><li> Expulsar o sangue a partir da agulha para uma área limpa de uma folha de parafilme (Figura 1K). </li><li> Medir glicose no sangue usando qualquer glicosímetro portátil comercial (Figura 1D). Nota: O glucometer usa um eléctrodo de flavina-adenina-dinucleótido-desidrogenase de glucose e exige um volume de amostra de 0,6 ul. <ol><li> Insira uma tira de teste completamente no medidor e directamente tocar a gota de sangue. Observe a coleta de sangue na tira de teste automaticamente e obter a glicose no sangue resultado 5 segundos na área de exibição. Anote o resultado e descartar a tira de teste. Use umnova tira de teste para cada medição. </li></ol></li><li> (passo facultativo) Leve quantidades precisas de sangue por uma pipetagem e transferir o sangue para um tubo de microcentrífuga para análises posteriores (hemoglobina, triacilglicerol (TG), colesterol total, etc.). Se necessário, diluir o sangue total de um peixe com solução salina. Centrifugar as amostras de sangue durante 3 min a 680 xg à temperatura ambiente e recolher o plasma. Transfira o plasma para um novo tubo. Neste ponto, que está pronta para ser utilizada em análises bioquímicas. </li></ol>

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The authors have nothing to disclose.

Nós apresentamos aqui um protocolo detalhado para a obtenção de sangue em série a partir de peixe-zebra adulto. Este método é simples de realizar e usá-lo no laboratório, numa base diária. Este método de coleta de sangue é baseada na inserção de uma agulha capilar de vidro em aorta dorsal do peixe-zebra. Durante este procedimento, é crítico para ter o cuidado de não fazer a ablação da coluna porque é o critério para a busca de aorta dorsal. Reduzir a lesão da coluna irá melhorar a taxa de sobreviv?...

Zebrafish estão ganhando cada vez mais popularidade como um valioso modelo de doenças humanas porque os seus órgãos e genética são semelhantes aos dos seres humanos 1,2. No campo da biologia do desenvolvimento, muitos estudos têm demonstrado que o peixe-zebra e mostrar humana marcada semelhança na hematopoiese 3, hemostasia 4,5, e 6 myelopoiesis. Peixe-zebra adulto também são usados ​​para o estudo imunológico 7, 8 e neurodegenerativa doenças relacionadas à obesidade 9 porque este ações organismo modelo vias comuns com aqueles interrompido em doenças humanas. Para obesidade e doenças relacionadas à obesidade (diabetes, esteatose hepática não alcoólica e e esteatohepatite aterosclerose), glicemia peixe-zebra e níveis de lipídeos, foram exaustivamente investigados em vários modelos de obesidade transgênicos e induzida por dieta 10-13. Coleta de sangue repetidas de animais individuais irá reduzir o uso de animais e dezembroRease diferenças interindividuais. No entanto, a recolha repetida de amostra é tecnicamente difícil em pequenos animais, tais como peixe-zebra por causa do seu volume de sangue relativamente pequena e a falta de vasos facilmente acessíveis. Vários métodos para a recolha de sangue de uma só vez a partir de peixes-zebra foram desenvolvidos, embora estes métodos têm os seus próprios inconvenientes, incluindo a letalidade, danos nos tecidos associados e volume de sangue limitado. Por exemplo, 1-5 mL de sangue pode ser colhido a partir de uma incisão lateral de aproximadamente 0,3 cm de comprimento na região da aorta dorsal 5. Decapitação com uma tesoura de cortar o cinto peitoral pode coletar 5-10 ul sangue 10. Outro método de amostragem de sangue conveniente é a ablação da cauda 14. Punção cardíaca é um potencial método alternativo para a coleta de sangue repetidas do mesmo peixe, mas a quantidade muito pequena obtida (aproximadamente 50 nl) com este procedimento limita o número de análises que podem ser performed 11. Por conseguinte, um novo protocolo é necessário para permitir a recolha de amostras de sangue não letal repetida, o que seria um avanço crítico necessário para este organismo ser um organismo modelo padrão para doenças humanas. Esta técnica permitiria testar resposta farmacológica, a descoberta de biomarcadores moleculares para o diagnóstico, determinação do prognóstico e do acompanhamento de várias doenças, tais como doenças metabólicas, doenças degenerativas e vários tipos de doenças malignas. Assim, desenvolvemos um método minimamente invasivo para a obtenção de sangue de peixe-zebra em série 15. Aqui demonstramos o procedimento visualmente e fornecer um protocolo detalhado para esta técnica. Usando este método, foram avaliados o valor normal com base em vários parâmetros, incluindo a hemoglobina, glicemia de jejum, e lipídios no sangue de peixe-zebra adultos saudáveis. Além disso, também avaliamos se esse método é apropriado para estudos que exigem amostras de série por mONITORIZAÇÃO as mudanças temporais nos níveis de glicose no sangue durante as experiências superalimentação.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass capillaries with filament</td>
			<td>Narishige</td>
			<td>GD-1</td>
			<td>1.0-mm-outer-diameter.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>To produce the needls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>081-00136</td>
			<td>For heparinization</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Drummond</td>
			<td>2-040-000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Bulb dispenser</td>
			<td>Drummond</td>
			<td>1-000-9000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-12075</td>
			<td>For anesthetizing the fish</td>
		</tr>
		<tr>
			<td>DRI-CHEM3500V</td>
			<td>Fujifilm</td>
			<td>-</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>DRI-CHEM Slides</td>
			<td>Fujifilm</td>
			<td>Hb-WII</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>Glutest Neo Super</td>
			<td>Sanwa Kagaku Kenkyusho</td>
			<td>-</td>
			<td>For bood glucose measurement</td>
		</tr>
		<tr>
			<td>Wako L-type TG kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>464-44201</td>
			<td>For TG measurement</td>
		</tr>
		<tr>
			<td>Wako L-type CHO kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>460-44301</td>
			<td>For total cholesterol measurement</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan Packaging</td>
			<td>PM996</td>
			<td>To expel the blood on</td>
		</tr>
	</tbody>
</table>

repeated blood collection for blood tests in adult zebrafish

Recolha de sangue é repetida uma das técnicas mais comuns realizados em animais de laboratório. No entanto, um protocolo não-letal para a recolha de sangue do peixe-zebra não foi estabelecida. Os métodos anteriores para a recolha de sangue a partir de peixe-zebra são letais, tais como incisão lateral, decapitação e ablação cauda. Assim, temos desenvolvido um novo método de recolha de sangue &quot;repetida&quot;, e aqui presente um protocolo detalhado descrevendo esse procedimento. Este método é minimamente invasivo e resulta em uma taxa de mortalidade muito baixo (2,3%) para peixe-zebra, permitindo, assim, a amostragem de sangue repetidas do mesmo indivíduo. O volume máximo de recolha de amostras de sangue é dependente do peso do corpo do peixe. O volume de sangue para a amostragem repetida em intervalos deve ser ≤0.4% de peso corporal por semana ou ≤1% a cada 2 semanas, que foram avaliadas por medições de hemoglobina no sangue. Além disso, a hemoglobina, glicemia de jejum, triglicerídeos plasma (TG) e total cníveis holesterol em peixe-zebra adulto masculino e feminino foram medidos. Nós também aplicaram esse método para investigar a desregulação do metabolismo da glicose na obesidade induzida por dieta. Este método de coleta de sangue permitirá que muitas aplicações, incluindo glicose e metabolismo lipídico e estudos hematológicos, o que aumentará o uso de peixe-zebra como um modelo de doença organismo humano.

Este método de coleta de sangue causa lesão mínima de peixe-zebra (a &lt;1mm punção; Figura 1J) e produz uma taxa de mortalidade muito baixa de 2,3%. Foi examinado o volume máximo de sangue que pode ser recolhido a partir de um único peixe e avaliada a relação com o seu peso corporal (Figura 3). Descobrimos que o volume de sangue coletado máximo foi linearmente correlacionado com o peso corporal (R = 0,813). O maior volume de sangue coletado de um peixe indivíduo (peso corpor...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

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 ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.5K visualizações.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Vídeo: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Coleta de sangue do caranguejo em ferradura americano, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Biologia

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Todo-mount Análise imuno-histoquímica para Vascularização embrionárias Limb pele: um sistema modelo para estudar Morfogênese Branching Vascular em Embrião

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

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 ...

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 ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Técnicas de Análise de vesículas extracelulares utilizando citometria de fluxo

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).