Verfahren zur in-vitro-Fertilisation und gynogenetic Diploiden wurden Zebrafisch von Charline Walker in George Streisinger Labor an der University of Oregon in den 1970er und 1980er Jahren entwickelt. Die hier beschriebene Methode ist unverändert von Charline die Protokolle, die verfügbar sind in voller Höhe in der Zebrafisch Book 3. Verfahren zur in-vitro-Fertilisation und gynogenetic Diploiden wurden Zebrafisch von Charline Walker in George Streisinger Labor an der University of Oregon in den 1970er und 1980er Jahren entwickelt. Die hier beschriebene Methode ist unverändert von Charline die Protokolle, die verfügbar sind in voller Höhe in der Zebrafisch Book 3.

Übersicht der frühen Druck Hinweis: Rezepte für viele der Reagenzien in diesem Protokoll wie Tricaine, Fisch Wasser und Hanks Kochsalzlösung verwendet sind online verfügbar in Kapitel 10 des Zebrafisch-Buch 3 ( <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ) <ol><li> Produce Embryonen durch künstliche Befruchtung mittels UV-inaktivierte Spermien. </li><li> Unmittelbar Übertragung der befruchteten Eizellen zu einem Glasfläschchen (mit einer Kunststoff-Schnappdeckel) bis zum Rand gefüllt mit Ei Wasser. Decken Sie die offene Ende des Röhrchens mit einer dünnen Schicht von Latex und rasten Sie die Oberseite (in die ein Loch geschnitten wurde) auf die Flasche über den Gummi. </li><li> Legen Sie die Flasche in eine hydraulische Presse und bei 1,4 min nach der Befruchtung zu erhöhen den Druck zu 8.000 psi (Beachten Sie, dass viele Maschinen kalibriert werden, um den Druck auf einem 2-Zoll-Durchmesser Kolben ausgeübt zu lesen, und Lesungen müssen psi umgewandelt werden) </li><li> Beginnend bei 6 min nach der Befruchtung allmählich den Druck über einen Zeitraum von 1 min. </li></ol> Detaillierte frühen Druck Verfahren <ol><li> Am Tag vor Experiment <ol style="list-style-type:lower-alpha;"><li> Am späten Nachmittag, zu trennen Männchen und Weibchen gequetscht werden. Alle alten Männchen kann verwendet werden, da er nicht beitragen wird genetisches Material sein. Frauen achten sollten groß und fett in den Bauch. Männer achten sollten gelb und spry. </li><li> Halten Sie Frauen und Männer getrennt über Nacht in Vorratstanks, wo sie leicht in den Morgen zugegriffen werden kann. </li></ol></li><li> Am Morgen des Experiments <ol style="list-style-type:lower-alpha;"><li> Bereiten Hanks Lösung, fügen Natriumbicarbonat. </li><li> Bringen Sie zum Fischen Einrichtung: Eiskübel, Hanks, Tricaine, Timer, Rohre für das Sammeln Spermien. </li><li> Verdünnen Tricaine in Fischwasser </li></ol></li><li> Sammeln Spermien <ol style="list-style-type:lower-alpha;"><li> Bereiten Sie eine klare Flasche Hanks-Lösung auf Eis. Measure ~ 50 ul pro Stecker auf gequetscht werden. </li><li> Anesthetize 3 oder 4 Männer in einer Zeit, durch Eintauchen in Tricaine. Sie können aus der Narkose aufgehoben werden mit einem Plastiklöffel. </li><li> Spülen in Fisch abspülen und &quot;feucht-trocken&quot; auf einem Papiertuch (sehr wichtig: Wasser aktiviert Spermien). </li><li> Berg Fisch in einem Schaum Halter unter einem Stereomikroskop bei geringer Vergrößerung mit Auflicht. Trennen Sie die Bauchflossen mit einer Pinzette und die Position einer Mikrokapillare bei der Eröffnung der Kloake. </li><li> Streicheln Sie die Seiten des Fisches vorsichtig, aber fest mit glatten (Millipore) Pinzette. </li><li> Da die milchige Sperma aus der Geschlechtsöffnung kommen, sammeln sie in die Mikrokapillare mit sanftem Sog. </li><li> Pool der Spermien von mehreren Männchen in eiskaltem Hanks Salzlösung. Sperm 5 bis 10 Männern ist ausreichend für die Befruchtung von mehreren hundert Eier. Sperma in kalten Hanks ist weiterhin Eier befruchten effizient für mehrere Stunden oder sogar Tage. &quot;Eyeball&quot; die Konzentration der Spermien, Sammeln genug, um eine trübe Suspension zu machen. </li></ol></li><li> UV inaktiviert Sperma <ol style="list-style-type:lower-alpha;"><li> Füllen Sie den Boden eines Glases Petrischale mit Eis. </li><li> Setzen Sie ein Uhrglas auf dem Eis in der Schüssel. </li><li> Mit einer Pasteur Pipette die Spermien aus dem Reagenzglas auf dem Uhrglas. Achten Sie darauf, wie viel der Spermien in die Pipette wie möglich zu erhalten, ohne dass sich keine Luftblasen mit ihm. Auch nicht-Blase die Spermien in dem Uhrglas, wie du bist vertrieben sie aus der Pipette. Expel Spermien in eine dünne, gleichmäßige Schicht auf Uhrglas. DISCARD dieser Pipette. </li><li> Legen Sie die Petrischale in eine UV-Vernetzer </li><li> Schalten Sie Vernetzer für 2 Minuten </li><li> Mit einem sauberen Pipette, entfernen Sie die Spermien aus dem Uhrglas und überträgt es auf ein Eppendorf-Röhrchen markiert UV Sperma und auf Eis stellen. </li></ol></li><li> Eiersammlung und in-vitro-Fertilisation <ol style="list-style-type:lower-alpha;"><li> Anesthetize drei oder vier Frauen in einer Zeit, in Tricaine. </li><li> Spülen in Fisch abspülen und &quot;feucht-trocken&quot; auf einem Papiertuch. Überschüssiges Wasser quillt die Eier und verhindern, dass die Befruchtung. </li><li> Legen Sie die Frau in einem 35 mm Petrischale aus Kunststoff und mit einem feuchten (nicht nassen) Finger, vorsichtig, aber fest auf den Bauch drücken. Wenn sie bereit ist, Eier zu legen, werden sie kommen aus ganz leicht. </li><li> Sammeln Sie die Eier mit einem Spatel und gibt die Frau zu Wasser. Gute Eier sind gelblich, durchscheinend Farbe, während Eier, die in der weiblichen blieb zu lange weiß und wässrig sind (siehe unten). Um zu gewährleisten, erhalten Sie ein gutes Eiern, sammeln sie während der ersten 2 Stunden nach die Lichter angehen in der Fisch-Anlage. Keep Eiern bedeckt, um ein Austrocknen zu verhindern. </li><li> Add 30-50 ul des UV Spermiensuspension, um die Eier, rühren Sie vorsichtig mit Pipettenspitze. </li><li> Unmittelbarly hinzuzufügen ca. 1 ml der Fische Wasser und Start-Timer. Eier links an dieser Stelle entwickeln Haploiden werden. </li></ol></li><li> . Frühe Pressure Hinweis: Schritte ag innerhalb von 90 Sekunden nach der Befruchtung vollzogen sein muss! <ol style="list-style-type:lower-alpha;"><li> Starte Zeit bei Moment, dass Eier durch die Zugabe von Wasser aktiviert werden </li><li> Warten Sie einen Moment, und fügen Sie mehr Wasser, wirbeln die Eier in die Mitte des Tellers. </li><li> Unter Anwendung eines Cut-off und poliert Pasteur Pipette die befruchteten Eier, um den Druck abgefüllt. </li><li> Wenn nötig, fügen Sie mehr Ei Wasser die Fläschchen, so dass sie fast überfüllt sind, so dass eine Perle von Wasser an der Lippe des Vials. </li><li> Legen Sie eine Gummi-Spitze über den Wulst aus Wasser und befestigen Sie die Kunststoff-Deckel über den Gummi. </li><li> Legen Sie die Durchstechflasche (n) den Kopf in den Druckzylinder, indem mehr Ei Wasser zum Nachfüllen des Zylinders. Legen Sie die oben auf dem Zylinder fest. </li><li> Setzen Sie den Zylinder in der Presse mit dem Stempel auf und gelten £ 8.000 / sq. in. </li><li> Lassen Sie den Zylinder in der Presse bis 6 min nach dem Zeitpunkt der Aktivierung (für eine Gesamtzeit von 4,5 min Druck). </li><li> Bei 6 min nach der Aktivierung, entfernen Sie den Zylinder aus der Presse, und entfernen Sie das Fläschchen aus dem Zylinder. Vorsichtig trocken die Fläschchen, um die Kühlung des Embryos zu verhindern. </li><li> Setzen Sie den Zylinder ab und füllen es mit Ei Wasser. </li><li> Bereiten Sie das nächste Fläschchen durch teilweises Füllen sie mit Ei Wasser als nötig. </li><li> Wiederholen Sie dies für alle Chargen von Eiern. </li></ol></li></ol> Hinweis: Der Zylinder kann bis zu 3 Fläschchen zu einer Zeit, so möchten Sie vielleicht die Befruchtung der Eier ein paar Minuten zu sehen, ob andere Frauen bereits in der Tricaine geben Eier zu verzögern. Dies wird dazu beitragen die Dinge zu beschleunigen, indem man mehrere Partien auf einmal. Denken Sie auch daran, wenn die Presse verwendet wird stellen Sie keine mehr Fische in den Tricaine, bis die Behandlung abgeschlossen ist. Fisch gestatten in der Narkose zu lange kann sie töten. Zweitens, wenn Sie Fisch Squeeze, während die Presse verwendet wird, und erhalten Sie Eier, können sie zu trocken, um durch die Zeit der Presse wieder verwendet werden kann lebensfähig.

<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>

Es ist wichtig, um sicherzustellen, dass Embryonen nach diesem Verfahren hergestellten wahre gynogenetic Diploiden sind. Als Kontrollen erzeugen eine Kupplung von normalen diploiden Eier (indem sie beiseite einige der Spermien ohne UV-Inaktivierung) und eine Kupplung von haploiden Embryonen (indem sie beiseite ein Gelege von Eiern mit UV Spermium befruchtet, ohne durch die EP-Verfahren). An 1 Tag nach der Befruchtung haploide Embryonen haben einen kurzen Körper Achse, unregelmäßige Gehirn und Somiten Morphologie. Hap...

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<td>Tricaine</td>
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<td>Sigma</td>
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<td>Watch glass</td>
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<td>French Press</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

Heterozygotie in diploiden Eukaryoten macht oft genetische Studien umständlich. Methoden, die lebensfähige homozygoten diploiden Nachkommen zu erzeugen direkt aus heterozygoten Weibchen ermöglichen F1 mutagenisierten Weibchen direkt für schädliche Mutationen gescreent werden in einem beschleunigten freuen genetische Bildschirm. Streisinger et al.<sup&gt; 1,2</sup&gt; Beschriebenen Verfahren zur Herstellung gynogenetic (homozygot) diploid Zebrafisch durch Aktivierung Zebrafisch-Eier mit UV-Licht-inaktivierten Spermien und verhindert dadurch entweder der zweiten meiotischen oder das erste zygotischen Zellteilung durch physikalische Behandlungen (Wärme oder Druck), dass deploymerize Mikrotubuli. Die &quot;early pressure&quot; (EP)-Methode blockiert die Meiose II, die kurz nach der Befruchtung auftritt. Die EP-Methode erzeugt einen hohen Anteil an lebensfähigen Embryonen, die zu fruchtbaren Erwachsenen beiderlei Geschlechts entfalten können. Das Verfahren erzeugt Embryonen, die homozygot in allen loci mit Ausnahme derjenigen, die von ihren Centromer durch Rekombination während der Meiose I. Homozygote Mutationen sind in EP Kupplungen zwischen 50% für Zentromer loci und weniger als 1% für Telomer-Loci entdeckt wurden voneinander getrennt sind. Diese Methode stammt wörtlich aus der Zebrafisch Buch reproduziert<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

Dies ist ein Verfahren zur Erzeugung gynogenetic diploid Zebrafischembryonen (Embryonen, deren einzige genetische Beitrag kommt von der Mutter) durch die Blockierung der zweiten Reifeteilung unmittelbar nach der Befruchtung mit UV-Licht-inaktivierten Sperma. EP Embryonen sind nicht vollständig homozygot durch Rekombination während der ersten meiotischen Teilung, aber sie sind homozygot überhaupt loci, die nicht von ihrem Zentromer wurden durch Rekombination getrennt.

Machen Gynogenetic Diploid Zebrafisch von Anfang Druck

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 Aufrufe.  University of Oregon. Dies ist ein Verfahren zur Erzeugung gynogenetic diploid Zebrafischembryonen (Embryonen, deren einzige genetische Beitrag kommt von der Mutter) durch die Blockierung der zweiten Reifeteilung unmittelbar nach der Befruchtung mit UV-Licht-inaktivierten Sperma. EP Embryonen sind nicht vollständig homozygot durch Rekombination während der ersten meiotischen Teilung, aber sie sind homozygot überhaupt loci, die nicht von ihrem Zentromer wurden durch Rekombination getrennt.

Serie: 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.

Live-Imaging der Zebrafisch embryonalen Gehirn durch konfokale Mikroskopie

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 ...

Forschung

Biologie

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.

Erstellen MR Imaging Kinderspiel - Pediatric Neuroimaging-Protokoll, Richtlinien und Verfahren

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.

Generieren Chimäre Zebrafischembryonen durch 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 ...

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.

Atmosphärische Molecular Imaging von biologischen Geweben und Biofilme durch LAESI Massenspektrometrie

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.

Direkte Analyse von Einzelzellen durch Massenspektrometrie bei Atmosphärendruck

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.

Imaging Glykane in Zebrafisch-Embryonen durch metabolische Markierung und Bioorthogonale Click-Chemie

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-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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.

Die Produktion von Haploid Zebrafischembryonen durch<em&gt; In-vitro-</em&gt; Düngung

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 Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

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