Métodos de fertilização in vitro e diplóides gynogenetic foram desenvolvidos para zebrafish por Charline Walker no laboratório de George Streisinger na Universidade de Oregon nos anos 1970 e 1980. O método descrito aqui não é alterado a partir de protocolos Charline, que estão disponíveis na íntegra em O Livro Zebrafish 3. Métodos de fertilização in vitro e diplóides gynogenetic foram desenvolvidos para zebrafish por Charline Walker no laboratório de George Streisinger na Universidade de Oregon nos anos 1970 e 1980. O método descrito aqui não é alterado a partir de protocolos Charline, que estão disponíveis na íntegra em O Livro Zebrafish 3.

Visão geral da pressão inicial Nota: receitas para muitos dos reagentes utilizados neste protocolo, como tricaina, peixes de água salina e Hanks estão disponíveis online no capítulo 10 do Livro Zebrafish 3 ( <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ) <ol><li> Produzir embriões por fertilização in vitro com esperma inativado UV. </li><li> Transferir imediatamente os ovos fertilizados em um frasco de vidro (com uma tampa de encaixe de plástico) cheia até a borda com água de ovo. Cobrir a extremidade aberta do frasco com uma folha fina de látex de borracha e encaixe a parte superior (em que um buraco foi cortado) para o frasco sobre a borracha. </li><li> Coloque o frasco em uma prensa hidráulica e em 1,4 min após a fertilização elevar a pressão a 8.000 psi (Note que pressiona muitos são calibrados para ler a pressão exercida sobre um pistão de diâmetro de 2 polegadas, e as leituras devem ser convertidos em psi) </li><li> Início em 6 min após a fertilização, gradualmente, liberar a pressão durante um período de 1 min. </li></ol> Procedimento de pressão detalhada início <ol><li> Dia antes experimento <ol style="list-style-type:lower-alpha;"><li> No final da tarde, os machos e fêmeas em separado para ser espremido. Qualquer homem velho pode ser usado desde que ele não vai estar contribuindo material genético. As fêmeas devem olhar grandes e gordura na barriga. Homens devem olhar amarelo e ágil. </li><li> Segure fêmeas e machos separadamente durante a noite nos reservatórios de retenção, onde podem ser facilmente acessados ​​pela manhã. </li></ol></li><li> Manhã de experiência <ol style="list-style-type:lower-alpha;"><li> Prepare a solução de Hanks, adicione bicarbonato de sódio. </li><li> Trazer para instalação de peixe: balde de gelo, Hanks, tricaina, timer, tubos para coleta de esperma. </li><li> Diluir em água tricaina peixes </li></ol></li><li> Coletar o esperma <ol style="list-style-type:lower-alpha;"><li> Prepare um frasco de solução de clara de Hanks no gelo. Medida ~ 50 ul por macho para ser espremido. </li><li> Anestesiar 3 ou 4 homens ao mesmo tempo por imersão em tricaina. Elas podem ser retiradas do anestésico com uma colher de plástico. </li><li> Enxágüe em água de peixes e blot &quot;úmido-seco&quot; em uma toalha de papel (muito importante: o esperma ativa água). </li><li> Montagem de peixes em um suporte de espuma sob um estereomicroscópio com ampliação baixa com epi-iluminação. Separar os nadadeiras pélvicas com uma pinça e uma posição microcapilar na abertura da cloaca. </li><li> Curso os lados dos peixes suavemente, mas firmemente com suave (Millipore) fórceps. </li><li> Como o esperma leitoso sair do poro genital, coletá-lo no microcapilar usando uma sucção suave. </li><li> Piscina do esperma de vários machos em solução salina gelada de Hank. Esperma dos machos 50-10 é adequado para a fertilização de várias centenas de ovos. Esperma no frio Hanks vai continuar a fertilizar ovos de forma eficiente por várias horas ou mesmo dias. &quot;Eyeball&quot; a concentração de espermatozóides, recolhendo o suficiente para fazer uma suspensão turva. </li></ol></li><li> UV inativar os espermatozóides <ol style="list-style-type:lower-alpha;"><li> Encha o fundo de uma placa de Petri de vidro com gelo. </li><li> Coloque um vidro de relógio em cima do gelo no prato. </li><li> Com uma pipeta Pasteur, transferir o esperma do tubo de ensaio para o vidro de relógio. Ter certeza de obter o máximo de esperma na pipeta possível sem ocupar as bolhas de ar com ele. Além disso, não bolha do esperma no vidro de relógio como você é expulsá-los da pipeta. Expelir esperma em uma camada fina e uniforme em vidro de relógio. Descarte este pipeta. </li><li> Coloque a placa de Petri em um UV cross-linker </li><li> Ligue reticulador por 2 minutos </li><li> Usando uma pipeta limpa, remova o esperma do vidro de relógio e transferir para um tubo eppendorf marcada Sperm UV e colocar no gelo. </li></ol></li><li> Coleta de ovos e fertilização in vitro <ol style="list-style-type:lower-alpha;"><li> Anestesiar três ou quatro mulheres em um momento em tricaina. </li><li> Enxágüe em água de peixes e blot &quot;úmido-seco&quot; em uma toalha de papel. O excesso de água vai inchar os ovos e evitar a fecundação. </li><li> Coloque a fêmea em um prato de plástico 35 milímetros de Petri e com umidade (não molhado) dedos, pressione suavemente mas com firmeza na barriga. Se ela está preparada para botar ovos, eles vão sair com bastante facilidade. </li><li> Recolher os ovos com uma espátula e retorne a fêmea para a água. Ovos são uma cor boa, amarelado translúcido, que os ovos que permaneceram na mulher por muito tempo são brancos e aguado (veja abaixo). Para garantir a obtenção de ovos bons, coletá-los durante as primeiras 2 horas após as luzes se acendem na instalação de peixe. Mantenha os ovos cobertos para evitar ressecamento. </li><li> Adicionar 3-50 mL da suspensão de espermatozóides UV para os ovos, mexa delicadamente com a ponta da pipeta. </li><li> Imediatoly adicionar cerca de 1 ml de água de peixe e iniciar o temporizador. Ovos deixados para desenvolver neste momento será haplóides. </li></ol></li><li> . Pressure início Nota: as etapas ag deve ser realizado dentro de 90 segundos da fecundação! <ol style="list-style-type:lower-alpha;"><li> Iniciar o temporizador no momento que os ovos são ativados pela adição de água </li><li> Aguarde alguns instantes e adicione mais água, os ovos redemoinho para o centro do prato. </li><li> Utilizando um cut-off e polido Pasteur pipeta, transferir os ovos fertilizados para os frascos de pressão. </li><li> Se necessário, adicione mais água ovo para os frascos de modo que eles estão quase transbordando, deixando uma gota de água no lábio dos frascos. </li><li> Coloque uma rolha de borracha sobre o filete de água e segura a tampa de plástico sobre a borracha. </li><li> Coloque o frasco (s) de cabeça para baixo no cilindro de pressão, adicionando uma água mais ovos para encher o cilindro. Coloque a parte superior do cilindro de forma segura. </li><li> Coloque o cilindro na imprensa com o êmbolo para cima e aplicar £ 8.000 / m². pol </li><li> Deixe o cilindro na imprensa até 6 min após o tempo de ativação (por um tempo total de 4,5 min de pressão). </li><li> Aos 6 min após a ativação, retire o cilindro da imprensa, e retire os frascos do cilindro. Cuidadosamente os frascos secos para evitar resfriamento dos embriões. </li><li> Redefinir o cilindro e recarregá-lo com água do ovo. </li><li> Prepare os frascos próxima preenchendo parcialmente com água de ovo, conforme necessário. </li><li> Repita o procedimento para todos os lotes de ovos. </li></ol></li></ol> NOTA: O cilindro irá realizar até 3 frascos de cada vez, então você pode querer atrasar a fecundação dos ovos de alguns minutos para ver se outras fêmeas já no tricaina dar ovos. Isso vai ajudar a acelerar as coisas, tratando lotes mais de uma vez. Além disso, lembro de uma vez a imprensa está sendo usado não colocar mais peixes no tricaina até que o tratamento seja concluído. Permitindo que os peixes para ficar no anestésico muito tempo pode matá-los. Em segundo lugar, se você apertar peixe, enquanto a imprensa está sendo usado, e obter os ovos, elas podem ser muito seco para ser viável no momento em que a imprensa pode ser usado novamente.

<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>

É importante certificar-se que os embriões produzidos por este método são diplóides gynogenetic verdade. Como controles, gerar uma ninhada de ovos diplóides normal (mantendo de lado algum do esperma sem UV inativação) e uma embreagem de embriões haplóides (mantendo de lado uma ninhada de ovos fertilizados com esperma UV sem passar pelo procedimento EP). Menos 1 dia após a fertilização de embriões haplóides têm um eixo de corpo curto, cérebro e morfologia irregular somito. Haplóides não são viáveis ?...

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making gynogenetic diploid zebrafish by early pressure

Heterozigosidade em eucariotos diplóides muitas vezes faz estudos genéticos pesado. Métodos que produzir descendentes viáveis ​​diplóide homozigoto diretamente de mulheres heterozigotas permitir que as fêmeas F1 mutagenizados para ser exibido diretamente para mutações deletérias em uma tela acelerado para frente genética. Streisinger et al.<sup&gt; 1,2</sup&gt; Descritos métodos para fazer gynogenetic zebrafish diplóide (homozigoto), ativando os ovos zebrafish com o esperma de luz ultravioleta e inativada prevenção, quer meiótica a segunda ou a primeira divisão celular zigóticos usando tratamentos físicos (calor ou pressão) que os microtúbulos deploymerize. A &quot;pressão inicial&quot; (EP) método bloqueia a meiose II, que ocorre logo após a fertilização. O método EP produz uma alta porcentagem de embriões viáveis ​​que podem se desenvolver para adultos fértil de ambos os sexos. O método gera embriões que são homozigotos em todos os loci, exceto aqueles que foram separados de suas centrômero por recombinação durante a meiose I. homozigótica mutações são detectadas em embreagens PE, entre 50% para loci centromérica e menos de 1% para loci telomérica. Este método é reproduzida na íntegra do livro 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

Este é um método para a geração de embriões diplóides gynogenetic zebrafish (embriões cuja única contribuição genética vem da mãe), bloqueando a segunda divisão meiótica imediatamente após a fertilização com esperma de luz ultravioleta inativado. EP embriões não são totalmente homozigótica devido à recombinação durante a primeira divisão meiótica, porém eles são homozigotos em todos os loci que não foram separadas de suas centrômero por recombinação.

Fazendo Zebrafish Diplóide Gynogenetic por pressão inicial

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

making-gynogenetic-diploid-zebrafish-by-early-pressure

Research

JoVE Journal

Biology

13.4K visualizações.  University of Oregon. Este é um método para a geração de embriões diplóides gynogenetic zebrafish (embriões cuja única contribuição genética vem da mãe), bloqueando a segunda divisão meiótica imediatamente após a fertilização com esperma de luz ultravioleta inativado. EP embriões não são totalmente homozigótica devido à recombinação durante a primeira divisão meiótica, porém eles são homozigotos em todos os loci que não foram separadas de suas centrômero por recom...

Vídeo: 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.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

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 ...

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.

Jogue Criança fazendo MR Imaging - Protocolo de neuroimagem Pediátrica, Diretrizes e Procedimento

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.

A criação de embriões quiméricos Zebrafish por Transplante

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.

Pressão atmosférica Imagem Molecular de tecidos biológicos e Biofilmes por LAESI Espectrometria de 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.

Análise direta de células individuais por Espectrometria de Massa à pressão atmosférica

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.

Glicanos imagem em embriões Zebrafish por Labeling Metabólica e Química Clique 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.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

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

In Vivo 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 Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

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.

Produção de Haplóide embriões Zebrafish por<em&gt; In Vitro</em&gt; Fertilização

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.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

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