Metodi per la fecondazione in vitro e diploidi gynogenetic sono stati sviluppati per zebrafish da Charline Walker nel laboratorio di Giorgio Streisinger presso l&#39;Università dell&#39;Oregon negli anni 1970 e 1980. Il metodo descritto qui è invariato rispetto a protocolli Charline, che sono disponibili per intero nel Libro Zebrafish 3. Metodi per la fecondazione in vitro e diploidi gynogenetic sono stati sviluppati per zebrafish da Charline Walker nel laboratorio di Giorgio Streisinger presso l&#39;Università dell&#39;Oregon negli anni 1970 e 1980. Il metodo descritto qui è invariato rispetto a protocolli Charline, che sono disponibili per intero nel Libro Zebrafish 3.

Panoramica di pressione all&#39;inizio Nota: le ricette per molti dei reagenti utilizzati in questo protocollo come tricaine, pesci d&#39;acqua salina e Hanks sono disponibili online nel capitolo 10 del Libro Zebrafish 3 ( <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ) <ol><li> Produrre embrioni da fecondazione in vitro con lo sperma UV inattivato. </li><li> Trasferire immediatamente le uova fecondate in un flacone di vetro (con tappo a scatto di plastica) riempiti fino all&#39;orlo di acqua uovo. Coprire l&#39;estremità aperta del flaconcino con un sottile foglio di lattice di gomma e far scattare la parte superiore (in cui un foro è stato tagliato) sulla fiala sopra la gomma. </li><li> Porre la fiala in una pressa idraulica e 1,4 minuti dopo la fecondazione aumentare la pressione a 8000 psi (Si noti che molte macchine sono tarati per leggere la pressione esercitata su un pistone da 2 pollici di diametro, e le letture devono essere convertiti in psi) </li><li> Con inizio alle 6 minuti dopo la fecondazione, rilasciare la pressione gradualmente in un periodo di 1 min. </li></ol> Dettagliata procedura di pressione all&#39;inizio <ol><li> Giorno precedente esperimento <ol style="list-style-type:lower-alpha;"><li> Nel tardo pomeriggio, separati maschi e femmine per essere spremuto. Qualsiasi maschio può essere utilizzato dal momento che non sarà contribuendo materiale genetico. Femmine dovrebbe apparire grandi e grassi nel ventre. I maschi dovrebbe apparire giallo e spry. </li><li> Tenere femmine e maschi separatamente durante la notte in serbatoi dove possono essere facilmente accessibili al mattino. </li></ol></li><li> Mattina di esperimento <ol style="list-style-type:lower-alpha;"><li> Preparare la soluzione Hanks, aggiungere bicarbonato di sodio. </li><li> Portare a struttura pesce: secchiello del ghiaccio, Hanks, Tricaine, timer, tubi per la raccolta di sperma. </li><li> Diluire Tricaine in acqua i pesci </li></ol></li><li> Raccogliere lo sperma <ol style="list-style-type:lower-alpha;"><li> Preparare una fiala di soluzione chiara Hanks sul ghiaccio. Misura circa 50 microlitri per ogni maschio a essere spremuto. </li><li> Anestetizzare 3 o 4 maschi in un momento di immersione in tricaine. Possono essere tirato fuori l&#39;anestetico con un cucchiaio di plastica. </li><li> Risciacquare con acqua e asciugare i pesci &quot;umido-secco&quot; su un tovagliolo di carta (molto importante: l&#39;acqua sperma attiva). </li><li> Monte di pesce in un supporto di schiuma sotto uno stereomicroscopio a basso ingrandimento con epi-illuminazione. Separare le pinne pelviche con una pinza e posizionare un microcapillare in apertura della cloaca. </li><li> Corsa ai lati del pesce delicatamente ma con fermezza, con liscio (Millipore) pinze. </li><li> Come lo sperma lattiginoso uscire dal poro genitale, si raccolgono nei microcapillare con aspirazione delicata. </li><li> Piscina allo sperma di maschi diversi in soluzione salina ghiacciata di Hank. Spermatozoi dei maschi 50-10 è adeguato per la fecondazione di diverse centinaia di uova. Spermatozoi nel freddo Hanks continuerà a fecondare le uova in modo efficiente per diverse ore o anche giorni. &quot;Eyeball&quot; la concentrazione di spermatozoi, raccogliendo abbastanza per fare una sospensione torbida. </li></ol></li><li> UV inattivare gli spermatozoi <ol style="list-style-type:lower-alpha;"><li> Riempire il fondo di un piatto di vetro Petri con ghiaccio. </li><li> Mettere un vetro d&#39;orologio in cima al ghiaccio nel piatto. </li><li> Utilizzando una pipetta Pasteur, trasferire lo sperma dalla provetta sul vetro d&#39;orologio. Essere sicuri di ottenere il maggior numero di spermatozoi nella pipetta possibile senza occupare eventuali bolle d&#39;aria con essa. Inoltre, non bolla il seme nel vetro d&#39;orologio, come li state espellendo dalla pipetta. Espellere lo sperma in un sottile e uniforme strato di vetro da orologio. Scarta questa pipetta. </li><li> Porre la capsula di Petri in un UV cross-linker </li><li> Accendere reticolante per 2 minuti </li><li> Utilizzando una pipetta pulita, togliere lo sperma dal vetro d&#39;orologio e trasferirlo ad una provetta Eppendorf segnato sperma UV e posto sul ghiaccio. </li></ol></li><li> Raccolta degli ovuli e la fecondazione in vitro <ol style="list-style-type:lower-alpha;"><li> Anestetizzare tre o quattro donne in un momento in tricaine. </li><li> Risciacquare con acqua e asciugare i pesci &quot;umido-secco&quot; su un tovagliolo di carta. L&#39;acqua in eccesso si gonfiano le uova e impedire la fecondazione. </li><li> Posizionare la femmina in un piatto di plastica 35 millimetri di Petri e umido (non bagnato) barrette, premere delicatamente ma con fermezza sul ventre. Se lei è pronto a deporre le uova, che verrà fuori abbastanza facilmente. </li><li> Raccogliere le uova con una spatola e tornare la donna in acqua. Le uova sono un buon giallo, colore traslucido, che le uova che sono rimasti nella femmina troppo a lungo sono di colore bianco e acquoso (vedi sotto). Per assicurarsi di andare uova buone, li raccolgono durante le prime 2 ore dopo che le luci si accendono nella struttura di pesce. Tenere le uova al coperto per evitare l&#39;essiccazione-out. </li><li> Aggiungere 30-50 ml di sospensione sperma UV per le uova, mescolate delicatamente con la punta della pipetta. </li><li> ImmediatoLy aggiungere circa 1 ml di acqua pesce e avviare il timer. Uova rimaste per lo sviluppo, a questo punto sarà haploids. </li></ol></li><li> . Pressione primi Nota: la procedura ag deve essere eseguita entro 90 secondi di fecondazione! <ol style="list-style-type:lower-alpha;"><li> Avvia timer momento che le uova vengono attivate con l&#39;aggiunta di acqua </li><li> Attendere qualche minuto e aggiungere altra acqua, agitare le uova al centro del piatto. </li><li> Usando un cut-off e lucidato pipetta Pasteur, trasferire le uova fecondate al fiale di pressione. </li><li> Se necessario, aggiungere acqua uovo di più per le fiale in modo che siano quasi straripante, lasciando una goccia d&#39;acqua a bordo della fiale. </li><li> Posizionare un tappo in gomma sulla goccia di acqua e fissare il coperchio di plastica sopra la gomma. </li><li> Posizionare il flaconcino (s) a testa in giù nel cilindro a pressione, aggiungendo acqua uovo di più per riempire il cilindro. Mettere la parte superiore del cilindro in modo sicuro. </li><li> Posizionare il cilindro nella stampa con lo stantuffo e applicare 8.000 £ / mq. in </li><li> Lasciare il cilindro nella stampa fino a 6 minuti dopo il tempo di attivazione (per un tempo totale di 4,5 min di pressione). </li><li> A 6 minuti dopo l&#39;attivazione, rimuovere il cilindro dalla stampa, e togliere i flaconcini dal cilindro. Asciugare con cura le fiale per evitare un raffreddamento degli embrioni. </li><li> Resettare il cilindro e riempirlo con l&#39;acqua uovo. </li><li> Preparare i flaconi parzialmente loro successivo riempimento con acqua uovo, se necessario. </li><li> Ripetere l&#39;operazione per tutte le partite di uova. </li></ol></li></ol> NOTA: Il cilindro può contenere fino a 3 flaconcini alla volta, quindi si consiglia di ritardare la fecondazione di uova qualche minuto per vedere se altre femmine già nel tricaine dare uova. Ciò contribuirà ad accelerare trattando più lotti allo stesso tempo. Inoltre, ricorda una volta che la stampa è in uso non inserire tutti i pesci più nel tricaine fino a quando il trattamento è stato completato. Permettendo di pesce di rimanere in anestesia troppo lungo può ucciderli. In secondo luogo, se si spremere i pesci, mentre la stampa è in uso, e ottenere le uova, possono essere troppo secca per essere valida al momento della stampa può essere riutilizzato.

<ol>
	<li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112 (2), 311-311 (1986).</li><li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-293 (1981).</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=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Johnson, S. L., Africa, D., Horne, S., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-tetrad+analysis+in+zebrafish:+mapping+the+ros+mutation+and+the+centromere+of+linkage+group+I.">Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.</a> Genetics. 139 (4), 1727-1727 (1995).</li><li>Beattie, C. E., Raible, D. W., Henion, P. D., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pressure+screens.">Early pressure screens.</a> Methods Cell Biol. 237 (71), 2575-25 (1999).</li><li>Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-linkage+analysis+and+consolidation+of+the+zebrafish+genetic+map.">Centromere-linkage analysis and consolidation of the zebrafish genetic map.</a> Genetics. 142 (4), 1277-1277 (1996).</li><li>Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutants+with+disrupted+early+T-cell+and+thymus+development+identified+in+early+pressure+screen.">Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.</a> Dev Dyn. 237 (9), 2575-2575 (1999).</li></ol>

E &#39;importante assicurarsi che gli embrioni prodotti con questo metodo sono diploidi gynogenetic vero. Come controlli, generano una frizione delle uova diploidi normali (tenendo da parte alcune delle spermatozoi senza UV-inattivazione) e una serie di embrioni aploidi (tenendo da parte una frizione delle uova fecondate con gli spermatozoi UV senza passare per la procedura di PE). A 1 giorno dopo la fecondazione degli embrioni aploidi hanno un asse corto del corpo, il cervello e la morfologia irregolare somite. Haploid...

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<td>Tricaine</td>
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<td>Pressure cylinder</td>
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<td>Instant ocean</td>
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<td>Pasteur pipettes</td>
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<td>Cut off the narrow end, fire polish</td>
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<td>UV cross-linker</td>
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making gynogenetic diploid zebrafish by early pressure

Eterozigosi negli eucarioti diploidi spesso rende studi genetici ingombrante. I metodi che producono prole vitale diploide omozigote direttamente dalle femmine eterozigoti permettono F1 femmine mutagenizzate di essere proiettati direttamente per le mutazioni deleterie in uno schermo accelerato in avanti genetica. Streisinger et al.<sup&gt; 1,2</sup&gt; Descritti metodi per rendere gynogenetic (omozigoti) zebrafish diploide attivando uova di pesce zebra con luce ultravioletta-inattivato sperma e la prevenzione o il meiotica seconda o prima divisione cellulare zigotico con trattamenti fisici (calore o pressione) che microtubuli deploymerize. La &quot;pressione precoce&quot; (EP) blocca il metodo meiosi II, che si verifica poco dopo la fecondazione. Il metodo PE produce un&#39;alta percentuale di embrioni vitali che possono sviluppare per adulti fertili di entrambi i sessi. Il metodo genera embrioni che sono omozigoti a tutti i loci ad eccezione di quelli che sono stati separati dai loro centromero dalla ricombinazione durante la meiosi I. mutazione omozigote vengono rilevati in frizioni tra il Parlamento europeo al 50% per i loci centromerica e meno dell&#39;1% per i loci telomerica. Questo metodo è riprodotto testualmente dal libro Zebrafish<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Making Gynogenetic Diploid Zebrafish by Early Pressure at JoVE.com

Making Gynogenetic Diploid Zebrafish by Early Pressure

Questo è un metodo per la generazione di embrioni di zebrafish gynogenetic diploide (embrioni il cui unico contributo genetico proviene dalla madre), bloccando la seconda divisione meiotica subito dopo la fecondazione con luce ultravioletta-inattivato sperma. Embrioni EP non sono completamente omozigoti per ricombinazione durante la prima divisione meiotica, tuttavia sono omozigoti a tutti i loci che non sono stati separati dalle loro centromero dalla ricombinazione.

Fare Diploide Zebrafish Gynogenetic di Pressure presto

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

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13.4K Views.  University of Oregon. Questo è un metodo per la generazione di embrioni di zebrafish gynogenetic diploide (embrioni il cui unico contributo genetico proviene dalla madre), bloccando la seconda divisione meiotica subito dopo la fecondazione con luce ultravioletta-inattivato sperma. Embrioni EP non sono completamente omozigoti per ricombinazione durante la prima divisione meiotica, tuttavia sono omozigoti a tutti i loci che non sono stati separati dalle loro centromero dalla ricombinazione.

Video: Making Gynogenetic Diploid Zebrafish by Early Pressure

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Ricerca

Biologia

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

Fare Giocare MR Child Imaging - Protocollo Neuroimaging Pediatrica, linee guida e procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

Generare embrioni di zebrafish Chimerico da trapianto

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Pressione atmosferica Imaging molecolare di tessuti biologici e biofilm da LAESI Spettrometria di Massa

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analisi diretta di singole cellule mediante spettrometria di massa a pressione atmosferica

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glicani Imaging in embrioni di zebrafish con marcatura metabolica e Chimica Clicca Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

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 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Produzione di Haploid Zebrafish embrioni da<em&gt; In Vitro</em&gt; Concimazione

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...