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 los animales procedimientos fueron aprobados por el Comité de Ética de la Universidad de Mie, y se realizaron de acuerdo a la regulación del bienestar animal japonesa &quot;Ley para el Bienestar y la Gestión de los Animales &#39;(Ministerio de Medio Ambiente de Japón) y cumplen con las directrices internacionales. 1. Preparación de la Aguja NOTA: Todos los experimentos se realizaron bajo anestesia, y se hicieron todos los esfuerzos para minimizar el sufrimiento. Para la eutanasia, los peces se sumergieron en un baño de agua con hielo (5 partes de hielo / 1 parte de agua a ≤4 ° C) durante ≥20 min. <ol><li> Preparar las agujas microcapilares vidrio tirando de un capilar de vidrio 1,0 mm de diámetro externo con un extractor de aguja (Figura 1A). </li><li> Cortar las puntas de las agujas de forma oblicua con unas tijeras finas (Figura 1B). El diámetro ideales punta debe ser de aproximadamente 100 a 200 micras (Figura 1C). Si el diamete puntar es demasiado estrecho, la sangre no va a entrar en la aguja. </li><li> Disolver la heparina en solución salina a una concentración de 5 mg / ml. </li><li> Coloque una aguja de precorte en el extremo revólver de un conjunto de tubo aspirador y mantenga la boquilla en la boca, o conecte la aguja a un dispensador bombilla. Sumerja la punta de la aguja en la solución de heparina y heparinizar la aguja de aspiración y sopla a través de la solución (Figura 1D y 1E). Nota: El conjunto de tubo aspirador se ha utilizado para la crioconservación de esperma de pez cebra 16. Tubo de goma largo puede detener cualquier sangre volando en la boca. Configuración de un filtro en la mitad de camino del tubo puede evitar los peligros. </li><li> Almacenar las agujas heparinizados en un plato de Petri de 10 cm y secar al aire durante al menos 1 hr. Un gran número de agujas se puede preparar con antelación. </li></ol> 2. Anestesia <ol><li> Preparar la solución anestésica en una pequeña caja de plástico mediante la mezcla de 200 ml de agua de pescado con 100 l de 2-fenoxietanol (2-EDUCACIÓN FÍSICA). La concentración final de los anestésicos es de 500 ppm. ¡CUIDADO! 2-PE tiene un efecto rápido. </li><li> Retire número deseado de pescado (cepa AB) desde el sistema de circulación. </li><li> Utilice una red para transferir el pescado en los anestésicos por 1 - 2 min (Figura 1F). Observar a los peces nadar gradualmente, difundir las aletas pectorales horizontalmente, jadeo, y tienen movimientos opérculo rápidos dentro de 1 min. </li><li> A medida que el tiempo pasa, observar los peces yacía en el fondo de la caja y, finalmente, dejar de nadar. El plano quirúrgico de anestesia alcanzará cuando los peces se detiene a jadear y los movimientos opérculo son lentos. En este punto, el pescado está listo para recogida de sangre. </li><li> El uso de un skimmer levantar el pescado anestesiado de la 2-PE y suavemente colóquela sobre una toalla de papel empapada con los anestésicos (Figura 1G). Cubra la cabeza del pescado con papel tisú suave también empapado con solución 2-PE para evitar la sequedad de los ojos y usar otro pañuelo de papel suave y seco para suavementesecar la superficie del cuerpo. </li></ol> 3. Blood Collection <ol><li> Coloque una aguja heparinizada al final revólver del conjunto del tubo de aspiración (o el dispensador bombilla) y mantenga el extremo de la boquilla del conjunto del tubo de aspiración en la boca. </li><li> Sujete el extremo boquilla y la aguja juntos, y retirar con cuidado las escalas que interfieren con la punta de la aguja. Inserte la aguja en un 30 - ángulo ° 45 en el sitio de la extracción de sangre. Evitar la punción del tracto gastrointestinal (Figura 1H). En caso de utilizar dispensador de bulbo, presione la bombilla con el pulgar y el dedo medio, y bloquear el agujero en la punta de la bombilla con el dedo índice, a continuación, inserte la aguja como se describe anteriormente. Nota: El sitio para la recogida de sangre es lo largo del eje del cuerpo y posterior al ano en la región de la aorta dorsal. La aorta dorsal (DA) y la vena cardinal posterior (PCV) son sólo ventral de la columna vertebral (Figura 2). </li><li> Comience a chupar lala boquilla extremo del conjunto de tubo de aspiración cuando se siente la aguja tocando la columna vertebral. Si la sangre no se eleva, mover la punta de la aguja de manera sutil con la mano para estimular el flujo de sangre. Tenga en cuenta que una vez que la sangre se está levantando en la aguja, para inmediatamente sacudidas y chupar suavemente (Figura 1I). En caso de utilizar dispensador de bulbo, liberar la presión de la bombilla para aspirar la sangre. </li><li> Observe la sangre se elevará lentamente en la aguja de una manera pulsátil sin aspiración, que es probablemente debido a la presión de la sangre arterial. Por lo tanto, no es necesario para aspirar si la aguja penetra correctamente la arteria. </li><li> Deja de succión después se recoge el volumen apropiado de la sangre. Retire la aguja de los peces y pulse el sitio de punción usando un pañuelo de papel suave para detener cualquier sangrado. Observar la parada de sangrado después de aproximadamente 10 a 20 segundos de la presión de los dedos (Figura 1J). </li><li> Después de que se detenga el sangrado, transferir inmediatamente el pescado de nuevo a un CLEuna de agua caliente (~ 28 ° C) tanque. Ayudar a los peces para recuperar girando suavemente agua hacia las branquias hasta que empiece a nadar. Nota: Mantenga hasta 5 postmuestreo peces en un tanque de 2 L y conectar el tanque para el sistema de circulación para oxigenar el pescado. La adición de antibióticos para el agua de pescado no es necesario. Mantener el pescado con una vivienda normal y la alimentación. </li><li> Expulsar a la sangre de la aguja en un área limpia de un trozo de parafina (Figura 1C). </li><li> Medir la glucosa en sangre usando cualquier glucómetro comercial de mano (Figura 1L). Nota: El glucómetro utiliza una adenina dinucleótido electrodo de deshidrogenasa de glucosa-flavina y requiere un volumen de muestra de 0,6 l. <ol><li> Inserte una tira reactiva por completo en el metro y tocar directamente la gota de sangre. Observe la extracción de sangre en la tira reactiva automáticamente y obtener la glucosa en la sangre número 5 seg en el área de visualización. Registre el resultado y deseche la tira reactiva. Usar unanueva tira de prueba para cada medición. </li></ol></li><li> (etapa opcional) Tomar cantidades precisas de sangre por un pipeteo y la transferencia de la sangre a un tubo de microcentrífuga para análisis posteriores (hemoglobina, triglicéridos (TG), colesterol total, etc.). Si es necesario, diluir la sangre entera de un pez con solución salina. Centrifugar las muestras de sangre durante 3 minutos a 680 xg a TA y cosechar el plasma. Transferir el plasma en un nuevo tubo. En este punto, está listo para ser utilizado en los análisis bioquímicos. </li></ol>

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The authors have nothing to disclose.

Presentamos aquí un protocolo detallado para la obtención seriada sangre de pez cebra adulto. Este método es sencillo de llevar a cabo y la usamos en el laboratorio en una base diaria. Este método de extracción de sangre se basa en la inserción de una aguja capilar de vidrio en aorta dorsal del pez cebra. Durante este procedimiento, es crítico que tener cuidado para no realizar la ablación de la columna vertebral debido a que es el criterio para la búsqueda de la aorta dorsal. La reducción de la lesión de la ...

El pez cebra están ganando cada vez mayor popularidad como un modelo valioso de enfermedades humanas debido a que sus órganos y la genética son similares a los de los humanos 1,2. En el campo de la biología del desarrollo, muchos estudios han demostrado que el pez cebra y el espectáculo humano marcados similitud en la hematopoyesis 3, hemostasia 4,5, y mielopoyesis 6. Pez cebra adulto también se utilizan para el estudio inmunológico 7, 8 y las enfermedades neurodegenerativas relacionadas con la obesidad 9 porque este organismo modelo comparte vías comunes con los perturbado en enfermedades humanas. Para la obesidad y las enfermedades relacionadas con la obesidad (diabetes, esteatosis hepática y la esteatohepatitis no alcohólica y aterosclerosis), la glucosa en sangre y los niveles de lípidos del pez cebra se han investigado a fondo en varios modelos de obesidad transgénicos y inducidos por la dieta 10-13. Muestreo de sangre repetida de animales individuales reducirá el uso de animales y diciembreRease diferencias interindividuales. Sin embargo, la recogida de muestras repetida es técnicamente difícil en animales pequeños, tales como el pez cebra debido a su relativamente pequeño volumen de sangre y la falta de vasos de fácil acceso. Varios métodos para la recogida de sangre de una sola vez de pez cebra se han desarrollado, aunque estos métodos tienen sus propios inconvenientes, incluyendo letalidad, daño tisular asociado y el volumen sanguíneo limitado. Por ejemplo, 1-5 de sangre l puede ser cosechado a partir de una incisión lateral de aproximadamente 0,3 cm de longitud en la región de la aorta dorsal 5. Decapitación con tijeras de corte a través de la cintura escapular puede cobrar 5-10 l de sangre 10. Otro método de muestreo de sangre conveniente es la ablación de la cola 14. Punción cardiaca es una potencial método alternativo para la recogida de sangre repetida de un mismo pescado, pero la cantidad muy pequeña obtenido (aproximadamente 50 nl) con este procedimiento limita el número de análisis que pueden ser PERFORmed 11. En consecuencia, se necesita un nuevo protocolo para permitir repetido muestreo de sangre no letal, que sería un avance crítica necesaria para este organismo para ser un organismo modelo estándar para enfermedades humanas. Esta técnica permitiría para el ensayo de respuesta farmacológica, el descubrimiento de biomarcadores moleculares para el diagnóstico, determinación del pronóstico y el seguimiento de diversas enfermedades, tales como enfermedades metabólicas, enfermedades degenerativas y varios tipos de tumores malignos. Por lo tanto, hemos desarrollado un método mínimamente invasivo para la obtención de sangre de pez cebra en serie 15. Aquí se demuestra el procedimiento visual y proporcionar un protocolo detallado para esta técnica. Usando este método, se evaluó el valor normal basado en diversos parámetros, incluyendo la hemoglobina, glucemia en ayunas y lípidos en la sangre de pez cebra adultos sanos. Además, también se evaluó si este método es adecuado para estudios que requieren muestras seriadas de mONITOREO los cambios temporales en los niveles de glucosa en la sangre durante los experimentos de sobrealimentación.

<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

Recogida de sangre repetida es una de las técnicas más comunes que se realizan en animales de laboratorio. Sin embargo, no se ha establecido un protocolo no letal para la recogida de sangre de pez cebra. Los métodos anteriores para la recogida de sangre de pez cebra son letales, como la incisión lateral, la decapitación y la ablación de la cola. Por lo tanto, hemos desarrollado un nuevo método de extracción de sangre &quot;repetida&quot;, y presentar aquí un protocolo detallado que describa este procedimiento. Este método es mínimamente invasiva y se traduce en una tasa de mortalidad muy baja (2,3%) para el pez cebra, lo que permite tomar muestras de sangre repetidas del mismo individuo. El volumen máximo de muestreo de sangre es dependiente sobre el peso corporal de los peces. El volumen para el muestreo de sangre repetida a intervalos debe ser ≤0.4% del peso corporal cada semana o ≤1% cada 2 semanas, que fueron evaluados mediante mediciones de la hemoglobina de la sangre. Además, la hemoglobina, glucemia en ayunas, triglicéridos en plasma (TG) yc totalesSe midieron los niveles holesterol en el pez cebra adultos masculinos y femeninos. También se aplica este método para investigar la desregulación del metabolismo de glucosa en la obesidad inducida por la dieta. Este método de extracción de sangre permitirá a muchas aplicaciones, incluyendo la glucosa y el metabolismo de los lípidos y los estudios hematológicos, lo que aumentará el uso de pez cebra como modelo de enfermedad organismo humano.

Este método de extracción de sangre causa daño mínimo para el pez cebra (a &lt;1 mm de punción; Figura 1J) y produce una tasa de mortalidad muy baja de 2,3%. Examinamos el máximo volumen de sangre que podría ser recolectada a partir de un solo pez y se evaluó la relación a su peso corporal (Figura 3). Se encontró que el volumen máximo de sangre recogida se correlaciona linealmente con el peso corporal (R = 0,813). El mayor volumen de sangre recogida de un pez individual (peso...

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.

Repetida extracción de sangre para pruebas de sangre en adultos de pez cebra

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

34.8K vistas.  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.

Video: 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.

Extracción de sangre del cangrejo herradura de América, 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 ...

Investigación

Biología

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.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

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 el montaje El análisis inmunohistoquímico de la piel del miembro Vasculatura embrionarias: un sistema modelo para estudiar la morfogénesis vascular ramificación en embriones

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.

En electroporación in vivo de Morpholinos en la retina de pez cebra adultos

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.

En electroporación in vivo de Morpholinos en la regeneración de adultos de pez cebra Aleta de cola

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.

Extracción de sangre para análisis bioquímicos en adultos de pez cebra

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 ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Un método práctico y novela para extraer el ADN genómico de los kits de extracción de sangre para la Preservación Plasma Protein

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

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.

Gavage adultos de pez cebra

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.

Un método para la detección y validación de mutaciones de resistencia contra la quinasa Inhibidores

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 para el Análisis de extracelular vesículas Usando citometría de flujo

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