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.

Tutte le procedure di animali sono stati approvati dal Comitato Etico dell&#39;Università di Mie, e sono state eseguite secondo normativa giapponese benessere degli animali &#39;legge sul Welfare e la gestione degli animali&#39; (Ministero dell&#39;Ambiente del Giappone) e il rispetto delle linee guida internazionali. 1. Preparazione del Needle NOTA: Tutti gli esperimenti sono stati eseguiti in anestesia, e sono stati fatti tutti gli sforzi per ridurre al minimo le sofferenze. Per l&#39;eutanasia, i pesci sono stati immersi in un bagno di acqua e ghiaccio (5 parti ghiaccio / 1 parte di acqua a ≤4 ° C) per ≥20 min. <ol><li> Preparare aghi microcapillari vetro tirando un capillare di vetro 1,0 mm di diametro esterno, con un estrattore ago (Figura 1A). </li><li> Tagliare le punte degli aghi obliquamente con le forbici sottili (Figura 1B). Il diametro della punta ideale dovrebbe essere di circa 100-200 micron (Figura 1C). Se la punta diameter è troppo stretta, il sangue non entrerà l&#39;ago. </li><li> Sciogliere eparina in soluzione salina ad una concentrazione di 5 mg / ml. </li><li> Inserire un ago pretagliato alla fine boccaglio di un gruppo del tubo aspiratore e tenere il boccaglio in bocca, oppure collegare l&#39;ago ad un erogatore lampadina. Immergere la punta dell&#39;ago nella soluzione di eparina, e eparinizzare l&#39;ago per aspirazione e soffia attraverso la soluzione (Figura 1D e 1E). Nota: Il gruppo del tubo di aspirazione è stato utilizzato per zebrafish crioconservazione degli spermatozoi 16. Lungo tubo di gomma in grado di fermare il sangue volare in bocca. Impostazione di un filtro nella metà del tubo può evitare i pericoli. </li><li> Conservare gli aghi eparinizzati in una capsula di Petri di 10 cm e asciugare per almeno 1 ora. Un gran numero di aghi può essere preparata in anticipo. </li></ol> 2. Anestesia <ol><li> Preparare la soluzione di anestetico in un piccolo caso di plastica mescolando 200 ml di acqua pesce con 100 ml di 2-fenossietanolo (2-PE). Concentrazione finale del anestetici è 500 ppm. ATTENZIONE! 2-PE ha un effetto rapido. </li><li> Rimuovere numero desiderato di pesce (ceppo AB) dal sistema di circolazione. </li><li> Utilizzare una rete per trasferire i pesci nelle anestetici per 1-2 min (Figura 1F). Osservare il pesce gradualmente nuotare, diffondere le pinne pettorali orizzontalmente, rantolo, e hanno movimenti rapidi opercolo entro 1 min. </li><li> Come il passare del tempo, osservare i pesci disteso sul fondo della cassa e finalmente smettere nuoto. Il piano di anestesia chirurgica raggiungerà quando il pesce si ferma a boccheggiare e movimenti opercolo sono lenti. A questo punto, il pesce è pronto per la raccolta di sangue. </li><li> Utilizzando uno skimmer sollevare il pesce anestetizzato dal 2-PE e delicatamente appoggiarlo su un tovagliolo di carta imbevuto di anestetici (Figura 1G). Coprire la testa del pesce con la carta dei tessuti molli anche imbevuto con una soluzione 2-PE per prevenire la secchezza degli occhi e utilizzare un&#39;altra carta velina morbido e asciutto per delicatamenteasciugare la superficie del corpo. </li></ol> 3. Il prelievo di sangue <ol><li> Mettere un ago eparinizzata alla fine revolver del gruppo del tubo aspiratore (o il distributore lampadina) e tenere l&#39;estremità portavoce del gruppo del tubo di aspirazione in bocca. </li><li> Afferrare l&#39;estremità revolver e l&#39;ago insieme, e rimuovere con attenzione le squame che interferiscono con la punta dell&#39;ago. Inserire l&#39;ago con un 30 - ° Angolo nel sito di raccolta del sangue 45. Evitare la puntura del tratto gastrointestinale (Figura 1 H). Nel caso di utilizzo di dispenser lampadina, premete la lampadina con pollice e il medio, e di bloccare il foro sulla punta del bulbo con il primo dito, poi inserire l&#39;ago come descritto sopra. Nota: Il sito per la raccolta del sangue è lungo l&#39;asse del corpo e posteriore all&#39;ano nella regione dell&#39;aorta dorsale. L&#39;aorta dorsale (DA) e la vena posteriori cardinale (PCV) sono solo ventrale al dorso (Figura 2). </li><li> Inizia a succhiare ilboccaglio estremità del gruppo del tubo di aspirazione quando l&#39;ago è sentita toccare la colonna vertebrale. Se il sangue non aumenta, spostare la punta dell&#39;ago sottilmente a mano per incoraggiare il flusso sanguigno. Si noti che una volta che il sangue è in aumento l&#39;ago, interrompere immediatamente agitazione e succhiare delicatamente (Figura 1I). In caso di utilizzo di dispenser bulbo, rilasciare la pressione del bulbo per aspirare il sangue. </li><li> Osservare il sangue aumentare lentamente l&#39;ago in modo pulsatile senza aspirazione, che è probabilmente a causa della pressione arteriosa. Pertanto, non è necessario per aspirare se l&#39;ago penetra correttamente l&#39;arteria. </li><li> Smettere di aspirazione dopo il volume appropriato di sangue viene raccolto. Rimuovere l&#39;ago dal pesce e premere il sito di puntura utilizzando carta velina morbido per fermare le emorragie. Osservare la fermata sanguinamento dopo circa 10-20 secondi di pressione delle dita (Figura 1J). </li><li> Dopo l&#39;emorragia viene arrestata, trasferire immediatamente il pesce di nuovo ad un CLEun riscaldamento dell&#39;acqua (~ 28 ° C) del serbatoio. Aiuta il pesce di recuperare rimestando delicatamente acqua verso le branchie fino a quando non comincia a nuotare. Nota: Mantenere fino a 5 postsampling pesci in una vasca 2 L e connessione del serbatoio al sistema di circolazione per ossigenare il pesce. L&#39;aggiunta di antibiotici all&#39;acqua pesce non è necessaria. Mantenere il pesce con custodia normale e l&#39;alimentazione. </li><li> Espellere il sangue dal dell&#39;ago su una superficie pulita di un pezzo di parafilm (Figura 1K). </li><li> Misurare la glicemia utilizzando qualsiasi glucometro portatile commerciale (Figura 1L). Nota: Il glucometro utilizza un glucosio-deidrogenasi flavina adenina dinucleotide elettrodo e richiede un volume del campione di 0,6 ml. <ol><li> Inserire una striscia reattiva completamente nello strumento e toccare direttamente la goccia di sangue. Osservare il prelievo di sangue nella striscia di test automatico e di ottenere il glucosio nel sangue risultato 5 secondi sul display. Registrare il risultato ed eliminare la striscia reattiva. Usa unnuova striscia reattiva per ogni misurazione. </li></ol></li><li> (passaggio facoltativo) Prendere quantità precise di sangue da un pipettaggio e trasferire il sangue in una provetta per ulteriori analisi (emoglobina, trigliceridi (TG), colesterolo totale, ecc.) Se necessario, diluire il sangue intero da un pesce con soluzione salina. Centrifugare i campioni di sangue per 3 min a 680 xg a temperatura ambiente e raccogliere il plasma. Trasferire il plasma in un nuovo tubo. A questo punto è pronto per essere utilizzato nelle analisi biochimiche. </li></ol>

<ol>
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The authors have nothing to disclose.

Vi presentiamo qui un protocollo dettagliato per l&#39;ottenimento in serie sangue di zebrafish adulto. Questo metodo è semplice da effettuare e lo usiamo in laboratorio su base giornaliera. Questo metodo di raccolta del sangue si basa sull&#39;inserimento di un capillare ago di vetro in aorta dorsale del pesce zebra. Durante questa procedura, è fondamentale stare attenti a non asportare la colonna vertebrale, perché è il criterio per la ricerca per l&#39;aorta dorsale. Ridurre il lesioni alla colonna vertebrale mig...

Zebrafish stanno guadagnando crescente popolarità come un modello valido di malattie umane perché i loro organi e genetica sono simili a quelli degli esseri umani 1,2. Nel campo della biologia dello sviluppo, molti studi hanno dimostrato che il pesce zebra e spettacolo umano contrassegnati somiglianza in ematopoiesi 3, emostasi 4,5, e mielopoiesi 6. Zebrafish adulti sono utilizzati anche per lo studio immunologico 7, 8 e le malattie neurodegenerative legate all&#39;obesità 9 perché questo modello azioni organismo percorsi comuni con quelli perturbato in malattie umane. Per l&#39;obesità e le malattie connesse con l&#39;obesità (diabete, steatosi epatica non alcolica e e aterosclerosi steatoepatite), zebrafish glucosio nel sangue e livelli di lipidi sono state investigate esaurientemente in vari modelli transgenici obesità e dieta-indotta 10-13. Ripetuto prelievo di sangue da singoli animali riduce il consumo animale e dicembrerease differenze interindividuali. Tuttavia, la raccolta dei campioni ripetuto è tecnicamente difficile in piccoli animali come zebrafish causa della loro relativamente piccolo volume di sangue e la mancanza di navi facilmente accessibili. Sono stati sviluppati diversi metodi per un tempo di raccolta del sangue da zebrafish, anche se questi metodi hanno i loro inconvenienti, tra cui letalità, danno tissutale associato e il volume di sangue limitata. Ad esempio, 1-5 microlitri di sangue può essere raccolto da un&#39;incisione laterale di circa 0,3 cm di lunghezza nella regione della aorta dorsale 5. Decapitazione con le forbici tagliando attraverso la cintura pettorale può raccogliere 5-10 microlitri di sangue 10. Un altro comodo metodo di campionamento del sangue è la coda di ablazione 14. Puntura cardiaca è una potenziale metodo alternativo per la raccolta di sangue ripetuto da uno stesso pesce, ma la piccola quantità ottenuta (circa 50 nl) con questa procedura limita il numero di analisi che possono essere effetmed 11. Di conseguenza, è necessario un nuovo protocollo per consentire ripetuto prelievo di sangue non letale, che sarebbe un progresso critica necessaria per questo organismo sia un organismo modello standard per le malattie umane. Questa tecnica consentirebbe di testare risposta farmacologica, la scoperta di biomarcatori molecolari per la diagnosi, la determinazione della prognosi e il monitoraggio di varie malattie, come le malattie metaboliche, malattie degenerative e diversi tipi di tumori maligni. Abbiamo quindi sviluppato un metodo minimamente invasivo per ottenere sangue da zebrafish in serie 15. Qui mostriamo la procedura visivamente e fornire un protocollo dettagliato per questa tecnica. Con questo metodo, il valore normale in base a vari parametri, tra cui l&#39;emoglobina, glicemia a digiuno, e lipidi nel sangue di zebrafish adulti sani sono stati valutati. Inoltre, abbiamo anche valutato se questo metodo è adatto per gli studi che richiedono campioni di serie di mONITORAGGIO i cambiamenti temporali nei livelli di glucosio nel sangue durante gli esperimenti sovralimentazione.

<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

Raccolta del sangue ripetuta è una delle tecniche più comuni eseguiti su animali da laboratorio. Tuttavia, non è stato stabilito un protocollo non letale per la raccolta del sangue da zebrafish. I metodi precedenti per la raccolta del sangue da zebrafish sono letali, come incisione laterale, decapitazione e la coda di ablazione. Così abbiamo sviluppato un metodo di raccolta del sangue &quot;ripetuto&quot; romanzo, e qui presenti un protocollo dettagliata che illustra questa procedura. Questo metodo è minimamente invasiva e comporta un tasso di mortalità molto basso (2,3%) per zebrafish, consentendo campionamento ripetuto sangue dello stesso individuo. Il volume massimo di prelievo di sangue dipende dal peso corporeo del pesce. Il volume per ripetuti prelievi di sangue a intervalli dovrebbe essere ≤0.4% del peso corporeo ogni settimana o ≤1% ogni 2 settimane, che sono stati valutati da misurazioni di emoglobina nel sangue. Inoltre, l&#39;emoglobina, glicemia a digiuno, trigliceridi del plasma (TG) e totale eclivelli holesterol in zebrafish adulti maschili e femminili sono stati misurati. Abbiamo applicato anche questo metodo per studiare la disregolazione del metabolismo del glucosio a obesità indotta dalla dieta. Questo metodo di raccolta del sangue consentirà numerose applicazioni, tra cui metabolismo glucidico e lipidico e gli studi ematologiche, che consentirà di aumentare l&#39;uso di zebrafish come un modello di malattia umana organismo.

Questo metodo di raccolta del sangue provoca lesioni minime di zebrafish (un &lt;1 millimetro puntura; Figura 1J) e produce un tasso di mortalità molto basso del 2,3%. Abbiamo esaminato il volume massimo di sangue che potrebbero essere raccolti da un singolo pesce e valutato il rapporto al peso corporeo (Figura 3). Abbiamo trovato che il volume massimo sangue raccolto è linearmente correlata con il peso corporeo (R = 0,813). Il maggior volume di sangue raccolto da un singolo pesce (pe...

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.

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

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

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

Research

JoVE Journal

Biology

34.5K Views.  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.

Raccolta di sangue dal Granchio Ferro di Cavallo Americano, Limulus Polifemo

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

Ricerca

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 la coltura di cellule endoteliali polmonari da topi neonati

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.

Con tutto il supporto per l&#39;analisi immunoistochimica vasi embrionali pelle degli arti: un sistema modello per studiare morfogenesi vascolare diramazione in embrione

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.

In elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

In elettroporazione in vivo di Morpholinos nella Adult Regenerating Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

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

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish adulti

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

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 metodo per lo screening e validazione di mutazioni resistenti contro 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 ...

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.

Tecniche per l&#39;Analisi dei extracellulare vescicole citometria a flusso

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).