Questi metodi sono stati sviluppati da EEL durante un congedo di ricerca nel laboratorio di JT è stato fornito supporto da parte dell&#39;Università DePaul Research Council e una NIH / NIDCR R01 concedere a JT (DE016678). Noi riconosciamo con gratitudine gli sforzi di Hunter Caroline, la cura degli animali tecnico nella struttura centrale zebrafish CMRC e Paulina Pawluczuk, il nostro assistente di laboratorio di laurea.

Zebrafish allevamento Zebrafish sono alloggiati e allevati secondo i metodi standard 1. Passato il periodo tardo larvale, lo stadio di sviluppo di un pesce zebra può essere misurata dalla sua lunghezza standard (SL), o la distanza retta in millimetri dal punto anteriormost del labbro superiore alla base della pinna caudale (posteriormost piastra hypural) 2. A circa un mese dopo la fecondazione (10-12 mm SL), i barbigli nasali e mascellari appaiono gemme epiteliali come trasparente vicino al box olfattive e sugli angoli posteriori dei mascellari, rispettivamente. Come il pesce zebra matura, il barbi si estendono in strette, baffi appendici simili. Perché il mascellare barbi è più grande (2-3 mm) e più facile da manipolare, il nostro protocollo si applica solo a questa appendice particolare; tecniche simili, tuttavia, potrebbero essere adattati alle più piccole la nasale barbo. Barbel Clipping <ol><li> Anestetizzare zebrafish adulti nella fase desiderato (ad es., 1,5-2,5 cm SL, ~ 3-6 mesi dopo la fecondazione) mediante immersione in 0.015% MS-222 (3-etil amminobenzoato metanosolfonato) tamponata a pH 7,0 in acqua sistema. Osservare i movimenti fino nuotare fermarsi e ventilazione branchie diventa lento e regolare. A seconda della dimensione, l&#39;anestesia completa dura circa 2-5 minuti. </li><li> L&#39;utilizzo di un acquario a rete, il trasferimento di pesce 1-3 alla volta per un pezzo piegato su un tovagliolo di carta umido in una capsula di Petri. Con una spatola smussato, orientare il pesce in modo che siano paralleli tra lato e laterale sinistro alto. Parte piega il tovagliolo di carta bagnato sulla parte caudale del pesce, che copre tutto il corpo fino alla opercolo (opercoli branchiali) per mantenere la pelle e le branchie umide durante la procedura. </li><li> Vista la piastra Petri con lo stereomicroscopio, con basso angolo di luce incidente ad illuminare la regione della testa. Focus sulla base della sinistra barbi mascellare, che sporge da un angolo posteriore ventrale del mascellare (Fig. 1). <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="Figura 1" /> Figura 1. </li><li> Afferrare saldamente l&#39;asta del distale leggermente verso il bordo della mascella barbo. Elevare l&#39;albero barbi e inserire le fauci di un punta fine forbici primavera appena prossimale al forcipe. Le forbici possono essere toccati per le pinze per la stabilità. Far scorrere le mascelle delle forbici lungo l&#39;asta del barbi fino a quando la punta di diamante si avvicina al bordo della mascella. Chiudere la forbice per tagliare l&#39;albero barbo. Il barbo amputato, ancora alle prese con pinza, può essere rimosso per la fissazione o ulteriori osservazioni. </li><li> Trasferire immediatamente il pesce a un piccolo serbatoio di acqua pulita del sistema (~ 500 ml) contenente una goccia di blu di metilene per il controllo superficiale infezioni fungine. Lasciare che il pesce di recuperare durante la notte. Il mattino seguente, ritorno il pesce al sistema di allevamento. Barbel rigenerazione è massima dopo 2 settimane. </li></ol> Incorporare agar di Matched Pairs Barbel <ol><li> Raccogliere i pesci con la tecnica appropriata e l&#39;eutanasia fissare in un tubo 50 ml di paraformaldeide al 4%-tampone fosfato (PBS) con agitazione a 4 ° C durante la notte. Il volume del fissativo deve essere almeno dieci volte il volume del tessuto. Dopo la fissazione, lo smaltimento dei rifiuti paraformaldeide in un contenitore approvato e lavare accuratamente il tessuto in diversi cambiamenti di PBS. </li><li> Preparare in un bicchiere da 250 mL pallone da 150 mL di agarosio al 2% (T m ~ 37 ° C) in acqua distillata. Calore con un forno a microonde o sulla piastra riscaldante, agitare periodicamente fino a completo scioglimento. Equilibrare la soluzione fuso in A ° C 50-60 bagnomaria. </li><li> Assemblare un impianto standard di elettroforesi su gel mettendo un piccolo stampo gel guarnizione (10 x 10 cm; ~ 100 volumi gel mL) saldamente contro i lati della camera di buffer. Aggiungere 2-4 piccoli pettini a denti campione (4 x 1,5 mm). </li><li> Su una superficie piana, versare l&#39;agarosio fuso nello stampo per coprire solo la base del pettine, formando pozzi molto superficiale. Subito messo l&#39;agarosio rimasto indietro nel bagno d&#39;acqua calda, che sarà utilizzato in seguito per integrare il tessuto (Piazza di 9-12). Metti una lunga pipetta Pasteur di vetro nel pallone per mantenere la pipetta alla stessa temperatura del agarosio. </li><li> Lasciare indurire il gel per 20-30 minuti. Rimuovere i favi, quindi rimuovere lo stampo gel contenente il gel indurito dalla camera buffer. Saldamente nastro i lati aperti dello stampo gel con del nastro di laboratorio in modo che il gel non scivolare fuori. Questo nastro dovrebbe estendersi diversi millimetri sopra la superficie del agarosio in modo che ulteriori agarosio può essere aggiunto in seguito (Step 13). </li><li> Posizionare lo stampo gel sul palco di uno stereomicroscopio. Ingrandire un pozzo vuoto e portare i bordi a fuoco. </li><li> Sulla superficie adiacente al pozzo agarosio, pipetta una goccia d&#39;acqua o tampone contenente i campioni fissati barbi (ad esempio, rigenerarsi e di controllo). USIng piegato perni insetti, trascinare i campioni umidi nel pozzo e spingerli verso il basso. Orientare il barbi alberi paralleli l&#39;uno all&#39;altro e allineati contro opposto lungo i bordi del pozzo. Tensione superficiale contribuirà a tenere il tessuto in posizione. </li><li> Quando si è pronti ad inserire, utilizzare una punta sottile pipetta o l&#39;angolo di un laboratorio di tessuti per rimuovere il liquido in eccesso dal pozzo. Lavorare velocemente per non lasciare che il tessuto asciugare. </li><li> Utilizzando la pipetta Pasteur in vetro riscaldato, delicatamente fresco pipetta agarosio fuso dentro e intorno i campioni per sigillare il tessuto barbi in posizione. Non riempire troppo. Prima l&#39;agarosio si indurisce, fare aggiustamenti dell&#39;ultimo minuto con i perni di insetti. </li><li> Inserire una etichetta numerata campione di carta accanto al pozzo e fissarlo con una piccola goccia di agarosio. </li><li> Ripetere i passaggi da 6 a 10 per ciascun pozzetto di campioni. </li><li> Se lo si desidera per la calibrazione di immagini, inserire un pezzo di carta con una scala micrometrica stampata (ad esempio, <a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a> ) nel fondo di un pozzo vuoto. Appiattire la carta sul fondo del pozzo e coprire con caldo agarosio. </li><li> Dopo che tutti i campioni sono incorporati, la superficie del gel sarà irregolare, con gocce indurite di agarosio. Posizionare il gel su una superficie piana e delicatamente versare ulteriori agarosio fuso fino a quando la superficie superiore è uniforme. Utilizzare la più piccola quantità di agarosio necessaria per ottenere una superficie liscia, troppo agarosio oscurerà a luce trasmessa fotografia. Permettere che questo strato superiore ad indurirsi. </li><li> Il gel può essere fotografato sul palco di uno stereomicroscopio per registrare morfologia lordi per ogni coppia barbo. Calibrazione immagine è ottenuta fotografando la scala micrometrica incorporato. </li><li> Il gel intero possono essere conservati avvolti in tovaglioli di carta bagnata e sigillato in un sacchetto di plastica a 4 ° per diverse settimane. </li><li> Per ulteriori analisi, blocchi di agar contenente coppia di barbigli può essere tagliato fuori dal gel con una lama di bisturi o un rasoio e trattati per istologia paraffina o criosezionamento con metodi standard di 3,4. In alternativa, barbi individuo può essere sezionato dalla agarosio con aghi sottili, lavate in acqua, e trattati per altre applicazioni a valle (ad esempio, con tutto il montaggio immunoistochimica e microscopia confocale). </li></ol>

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1+General+Methods+for+Zebrafish+Care.">Chapter 1 General Methods for Zebrafish Care.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Howe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+length:+Not+quite+so+standard.">Standard length: Not quite so standard.</a> Fisheries Research (Amsterdam). 56, 1-7 (2002).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+Tissue+Samples+for+Sectioning.">Paraffin Embedding Tissue Samples for Sectioning.</a> Cold Spring Harbor Protocols. 6, (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+8.3+Agar+Embedding+for+Cryostat+Sectioning+of+Embryos+or+Larvae.">Ch 8.3 Agar Embedding for Cryostat Sectioning of Embryos or Larvae.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Hansen, A., Reutter, K., Zeiske, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+development+in+the+zebrafish,+Danio+rerio.">Taste bud development in the zebrafish, Danio rerio.</a> Dev Dyn. 223, 483-496 (2002).</li></ol>

Il pesce zebra mascellare barbi è un sistema di tessuto sottoutilizzate per lo studio della crescita, la manutenzione e la rigenerazione di diversi tipi di cellule in zebrafish. Anche se l&#39;appendice barbi non ha l&#39;analogo umano, i tipi di cellule in esso contenute sono altamente conservati, rendendo possibile lo studio della pelle, ghiandole, melanociti, vasi circolatori e nervi in ​​una struttura otticamente chiara e anatomicamente semplice forma cilindrica. Simile al ben studiato pinna caudale, il tessuto barbi possono essere indotte a rigenerare da amputazione. Utilizzando il bordo della mascella come un punto di riferimento anatomico, il piano di amputazione può essere collocato con precisione, agevolare la misurazione dei barbi ricrescita. Per un operatore esperto, ogni intervento chirurgico richiede solo pochi secondi. Il recupero è rapido, e finora abbiamo rilevato alcun effetto a breve oa lungo termine sul comportamento dei pesci. Zebrafish con una nuotata mascellare barbi, mangiano e si riproducono nel modo più efficace non chirurgica controlli, e hanno la sopravvivenza paragonabile al momento della raccolta dei tessuti, fino a 6 mesi dopo l&#39;intervento chirurgico. L&#39;impatto fisiologico di questo intervento chirurgico previsto per essere minimo, perché 1) le papille gustative extraorali trasportati sulla barbi si trovano anche in molte altre parti del dell&#39;epitelio pesci, tra cui le labbra, le guance e la testa 5, e 2) papille gustative di differenziazione appaiono il barbo rigenerante entro 72 ore (LeClair et al., dati non pubblicati). Questo rende il mascellare barbi un sistema mini-invasivo per studiare la guarigione delle ferite, rivascolarizzazione, e reinnervazione nel contesto di un vertebrato adulto. Dopo l&#39;induzione chirurgica della rigenerazione, barbi possono essere raccolti ad intervalli per la misurazione morfometrica e / o l&#39;analisi microscopica dei tessuti fissi. Per comodità, il mascellare barbi è circa la lunghezza (2-3 mm) e diametro (100-200 mm) di un embrione di pesce zebra, facilitando l&#39;applicazione di molti protocolli standard, tra cui l&#39;istologia paraffina, criosezionamento, con tutto il montaggio immunoistochimica, ed in situ ibridazione. Prese insieme, queste caratteristiche rendono il mascellare barbi altamente fattibile modello in vivo per studiare la riparazione dei tessuti e la rigenerazione.

Microscopio Attrezzature Un microscopio stereo con dissezione trasmessa e incidente in fibra ottica di illuminazione è necessaria per clipping barbi e l&#39;incorporamento di agar. Barbel Clipping <ul><li> Pesce sistema idrico </li><li> 2 zebrafish incrocio serbatoi </li><li> MS-222 (3-etil amminobenzoato metanosolfonato; chimica Sigma # E10521-10G) </li><li> sterili da 120 mm piastra Petri </li><li> tovaglioli di carta bagnati </li><li> spatola metallica smussata </li><li> Dumont # 55 Bio Inox Pinza (Strumenti Scienza Belle # 11255-20) </li><li> Mini-Vännäs molla forbici (Strumenti Scienza Belle # 15000-00) </li><li> blu di metilene agente antimicotico (Dott. Foster &amp; Smith # CD-905781) </li><li> piccolo acquario a rete </li></ul> Raccolta dei tessuti e l&#39;incorporamento di agar <ul><li> Paraformaldeide 4% in 1x PBS (PF-PBS), fino aliquotati in 50 mL conica e congelati a -20 ° C usare </li><li> 1x PBS (pH 7,27-6) </li><li> 250 ml di vetro fiasco </li><li> Agarosio al 2% in acqua distillata </li><li> standard di piccole gel elettroforesi su rig (dimensioni del gel = ~ 10 cm quadrati) </li><li> piccolo campione pettine a denti elettroforesi (pettine formato 4 x 1,5 mm) </li><li> acqua bagno caldo (50-60 ° C) </li><li> laboratorio nastro </li><li> nero smaltato perni insetti, dimensioni 000 (Strumenti di scienze Belle # 26000-25) </li><li> 1-2 titolari pin (strumenti Scienza Belle # 26016-12) </li><li> Da 9 pollici in vetro pipetta Pasteur </li><li> pipetta a bulbo o della pompa </li><li> laboratorio di tessuti </li><li> etichette di carta di piccole </li><li> (Opzionale) scala di calibrazione stampato </li><li> tovaglioli di carta bagnati </li><li> zip di chiusura sacchetto di plastica </li><li> bisturi / lama di rasoio (per tagliare blocchi di agar) </li></ul>

methods for the study of the zebrafish maxillary barbel

Barbi sono annessi cutanei sensoriali si trovano in pesci, rettili e anfibi. Il pesce zebra, Danio rerio, sviluppa due paia di barbigli: un paio nasale corta e un paio più mascellare. Barbel tessuto contiene cellule di origine ectodermica, e l&#39;origine mesodermica cresta neurale, incluse le cellule della pelle, ghiandole, papille gustative, melanociti, vasi circolatori e nervi sensoriali. Diversamente dalla maggior parte dei tessuti adulti, il mascellare barbi è otticamente chiaro, che ci permette di visualizzare lo sviluppo e la manutenzione di questi tipi di tessuto per tutto il ciclo di vita. Questo video mostra lo sviluppo iniziale del mascellare barbi (inizio circa un mese dopo la fecondazione) e dimostra un protocollo chirurgico per indurre la rigenerazione in appendice adulti (&gt; 3 mesi dopo la fecondazione). In breve, il barbo mascellare sinistro di un pesce anestetizzato è elevata con una pinza sterile, appena distalmente al bordo caudale della mascella. Un bene, forbici sterili primavera è posizionato contro la pinza per tagliare l&#39;albero barbi a questo livello, che istituisce un punto di riferimento anatomico per il piano di amputazione. Crescita rigenerativa può essere misurata rispetto a questo piano, e rispetto al controlaterale barbo. Tessuto Barbel rigenera rapidamente, raggiungendo la ricrescita massimo entro 2 settimane dalla lesione. Tecniche di analisi del rigenerato barbi includono la dissezione e l&#39;incorporamento di coppie di barbigli (rigenerare e controllo) nei pozzetti di un gel elettroforesi standard di DNA. I campioni sono opportunamente integrati fotografato allo stereomicroscopio per la morfologia e la morfometria lordo, e può essere conservato per settimane prima di applicazioni a valle come istologia paraffina, criosezionamento, e / o immunoistochimica montare tutto. Questi metodi stabilire il barbi mascellare come un romanzo nel sistema del tessuto in vivo per lo studio della capacità rigenerativa di più tipi di cellule nel contesto genetico di zebrafish.

Watch this Scientific Journal Video about Methods for the Study of the Zebrafish Maxillary Barbel at JoVE.com

Methods for the Study of the Zebrafish Maxillary Barbel

Il pesce zebra mascellare barbi è un organo di senso tegumentario contenente ectodermica, mesodermica e derivati ​​della cresta neurale. È importante sottolineare che il barbo adulti in grado di rigenerare dopo l&#39;amputazione prossimale. Questo video presenta mascellare sviluppo barbi e dimostra un protocollo chirurgico per indurre la rigenerazione, seguita dalla raccolta di immagini, l&#39;incorporamento ea valle dei campioni barbo.

Metodi per lo studio del mascellare Zebrafish Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

methods-for-the-study-of-the-zebrafish-maxillary-barbel

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K Views.  DePaul University. Il pesce zebra mascellare barbi è un organo di senso tegumentario contenente ectodermica, mesodermica e derivati ​​della cresta neurale. È importante sottolineare che il barbo adulti in grado di rigenerare dopo l'amputazione prossimale. Questo video presenta mascellare sviluppo barbi e dimostra un protocollo chirurgico per indurre la rigenerazione, seguita dalla raccolta di immagini, l'incorporamento ea valle dei campioni barbo.

Video: Methods for the Study of the Zebrafish Maxillary Barbel

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated modello animale Stimoli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Utilizzando la risposta optocinetico per studiare la funzione visiva del pesce zebra

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Iniezione intraperitoneale in Zebrafish adulti

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Ablazione laser del Pronephros Zebrafish allo Studio renale rigenerazione epiteliale

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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 ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Metodi per l&#39;estrazione di Endosymbionts dalla Whitefly<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...