Name: affinity labeling detection of endogenous receptors from zebrafish embryos
Uploaded: 2016-08-31T15:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

Alle Versuche an Tieren durchgeführt wurden vom Ausschuss für Laboratory Animal Care und Verwenden der Autonomen Nationalen Universität von Mexiko (UNAM) genehmigt, unter dem CICUAL-Protokollnummer: FLC40-14. (CICUAL: &quot;Comité Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de fisiologia Celular, Universidad Nacional Autónoma de México&quot;). 1. Herstellung von Embryo Protein Extract <ol><li> Sammeln Sie 100 bis 200 Embryonen für jede Bedingung zu vergleichen (morphants vs. Wildtyp) in der gewünschten Stufe, zum Beispiel 72 Stunden nach der Befruchtung (HPF). </li><li> Platz Embryonen in Petrischalen mit Fischwasser (siehe Tabelle 1) und manuell dechorionate Embryonen mit feinen bestückte Zange. Während dieses Schritts zu vermeiden (oder einer anderen Protease gesamten Protokoll) unter Verwendung von Pronase, da es die gezielte Rezeptor verdauen kann. </li><li> Ort Embryonen in einem 1,5 ml Mikrozentrifugenröhrchen und wasche Embryonen twEis mit 1x Phosphatpufferlösung (PBS) (siehe Tabelle 1). </li><li> In 500 ul deyolk Puffer (siehe Tabelle 1). </li><li> Lassen Sie die meisten Eigelb durch vorsichtiges Pipettieren nach oben und unten (ca. 30 - 40-mal) die Embryonen in der Lösung. Für 72 HPF und 48 HPF Embryonen verwenden, blaue und gelbe Pipettenspitzen sind. Gelb Spitzen müssen leicht geschnitten werden kann, Zerstörung von Embryonen zu vermeiden. Die erfolgreiche Dotter Freisetzung kann durch die Beobachtung Embryonen unter dem Mikroskop geprüft werden. </li><li> Sammeln Embryonen durch Zentrifugation bei 600 xg für 15 sec. </li><li> Überstand verwerfen sorgfältig durch eine Pipette. </li><li> Wash Embryonen zweimal mit 500 &amp; mgr; l Waschpuffer (siehe Tabelle 1) durch bei niedrigster Geschwindigkeit sanft verwirbelt. </li><li> Sammeln Embryonen durch Zentrifugation bei 600 xg für 15 sec. </li><li> Ab hier geht das Verfahren bei 4 ° C. </li><li> Überstand verwerfen sorgfältig durch eine Pipette. </li><li> Resuspendieren Embryonen in 350 &amp; mgr; l Lysepuffer (siehe Tabelle 1) und homogenisieren einen Plastik zerstoßen. </li><li> Inkubieren lysiert Embryonen 30 min unter Rühren auf einem Reagenzglas rocker bei 4 ° C. </li><li> Zentrifuge lysiert Embryonen bei 11.000 xg für 15 min, um unlösliche Rückstände zu entfernen. </li><li> Transfer geklärte Überstand einer Pipette in ein frisches Röhrchen verwenden. </li><li> Bestimmung der Gesamtprotein durch Bradford-Test 4 oder ein anderes geeignetes Verfahren. Wenn Bradford - Test verwendet wird, führen Sie die Kalibrierungskurve in Gegenwart von äquivalenten Konzentrationen der Waschmittel präsentiert im Lysepuffer, da sie eine Unterschätzung des Proteins Standard - 4 führen. </li></ol> 2. Nachweis von endogenen Rezeptor Protein <ol><li> Affinitätsmarkierung und Immunpräzipitation (alle diese Schritte wurden bei 4 ° C) <ol><li> Platzieren 400-500 ug Gesamt - Embryo - Protein in einem 1,5 ml Mikrozentrifugenröhrchen und mit Wasser auf 1 &amp; mgr; g / &amp; mgr; l mit Puffer 1 (siehe Tabelle 1). Hinweis: Um 500 ug Gesamt-Embryo-Protein zu erhalten, etwa 100 bis 200 Embryonen müssen zunächst verarbeitet werden. 72 HPF Embryonen routinemäßig ergeben ~ 5 ug Gesamt-Embryo-Protein, das für Betaglycan Nachweis ausreichend ist. </li><li> Hinzufügen genügend Volumen an markiertem TGF-β2 Lager um eine Endkonzentration von 150 pM zu erreichen und inkubiere unter Schütteln auf einem Reagenzglas Wippe 2 h bei 4 ° C. TGF-β2 müssen gekennzeichnet werden , bevor AFLIP gestartet wird, mit 125 I nach dem Verfahren Chloramin T , wie durch Cheifetz et al. 5. ACHTUNG: Verwenden Sie Abschirmung Exposition zu minimieren , während 125 Umgang mit der Bezeichnung I - Liganden. </li><li> Hinzufügen genügend Volumen an unverdünntem Antikörper gegen den Rezeptor von Interesse, um eine 1 zu erreichen: 100 Verdünnung und weiter Inkubation für weitere 2 h bei 4 ° C (Inkubation kann O / N sein). Dies ist die optimale Verdünnung des Antiserums # 31, die in dieser Studie verwendet und anderswo 3 beschrieben, jedoch in Abhängigkeit von der Qualität der eintibody, eine größere oder kleinere Verdünnung kann die optimal sein. </li><li> Zugabe von 50 &amp; mgr; l geeignete Immunglobulin-Bindungsperlen (zB G-Protein-Sepharose, die zuvor in TNTE äquilibriert wurde und resuspendiert 1: 5 des ursprünglichen Volumens in TNTE) und Inkubation für 50 min unter Rühren auf einem Reagenzglas rocker bei 4 ° C. </li><li> Gewinnen Sie die Perlen durch Mikrozentrifugieren bei 11.000 × g für 20 Sekunden. </li><li> Überstand verwerfen in einem geeigneten radioaktiven Abfallbehälter. </li><li> Waschen Sie die IP-Perlen dreimal mit 1 ml IP - Waschpuffer (siehe Tabelle 1) durch Vortexen und Mikrozentrifugation bei 11.000 xg für 20 sec jedes Mal. </li><li> Resuspendieren IP-Perlen in 250 ul IP Waschpuffer. </li><li> Fügen 1,5 ul Disuccinimidylsuberat (DSS, gelöst in DMSO bei 10 mg / ml). Bereiten Sie die DSS-Lösung kurz vor der Verwendung in diesem Schritt. </li><li> Inkubieren für 15 min bei 4 ° C unter Rühren. </li><li> Um die Vernetzungsreaktion zu löschen, 500 &amp; mgr; l of IP Waschpuffer, ergänzt mit genügend Tris-Cl pH 7,4 Lager 25 mM Tris-Cl zu erreichen. Die freien Aminogruppen in Tris erfassen nicht umgesetzten DSS. </li><li> Zentrifuge bei 11.000 g für 20 sec IP-Perlen zu sammeln und Überstand verwerfen. </li><li> Resuspendieren IP-Perlen in 30 ul Puffer Laemmli reduzieren. </li><li> Boil Proben 5 min bei 94 ° C. </li><li> Optional analysieren Proben in einem Gamma-Zähler des Protokolls Hersteller. </li></ol></li><li> Probenanalyse <ol><li> Gegenstand Proben einer SDS-PAGE denaturieren. Verwenden Sie Polyacrylamid auf der entsprechenden Prozentsatz für die Masse des Rezeptors und führen Gel unter Standardverfahren. </li><li> Tauchen Gel in Fixierlösung (siehe Tabelle 1) für 30 min bei RT. </li><li> Waschen Sie Gel mit destilliertem Wasser 15 min. </li><li> Setzen Sie Gel in vorher hydratisiert Filterpapier und Deckel mit Saran Wrap Folie. </li><li> Trockenen Gels bei 80 ° C für 1 Stunde. </li><li> Expose Gel auf einem weißen Phosporimager screen bei RT O / N. </li><li> Scan ausgesetzt Bildschirm mit einem Phosporimager mit Protokoll des Herstellers. </li></ol></li></ol>

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The authors have nothing to disclose.

Die Verwendung von Western - Blots mit einem spezifischen Antikörper gegen ein Protein von Interesse ist ein wertvolles Instrument zum Ausdruck 7 während der Embryonalentwicklung zu studieren. Jedoch Immunoblotting hoch glycosylierten Proteinen war nicht sehr erfolgreich gewesen aufgrund ihrer ineffizienten Übertragung und eine schwache Bindung an Nitrocellulose oder PVDF Membranen 8,9. Proteoglykane sind ein gutes Beispiel für dieses Manko, die aufgrund ihrer koval...

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

<table><tbody><tr><td>Disuccinimidyl suberate (DSS)</td><td>ThermoFisher Scientific</td><td>21555</td><td></td></tr><tr><td>Protein G Sepharose 4 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0618-01</td><td></td></tr><tr><td>Gel Dryer Model 583&amp;nbsp;</td><td>BIO-RAD</td><td>1651745</td><td></td></tr><tr><td>Typhoon 9400</td><td>GE Healthcare Life Sciences</td><td>63-0055-78</td><td></td></tr><tr><td>Cobra II Auto gamma counter</td><td>Packard</td><td></td><td></td></tr><tr><td>Exposure Cassette</td><td>Molecular Dynamics</td><td>63-0035-44</td><td></td></tr><tr><td>NaCl</td><td>J.T. Baker</td><td>3624</td><td></td></tr><tr><td>KCl</td><td>J.T. Baker</td><td>3040</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3828</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3246</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;OH</td><td>J.T. Baker</td><td>9070</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;COOH</td><td>J.T. Baker</td><td>9508</td><td></td></tr><tr><td>HCHO</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>ED</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Crystal Sea Marine Mix</td><td>Marine Enterprises International</td><td>http://www.meisalt.com/Crystal-Sea-Marinemix</td><td></td></tr></tbody></table>

affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

Figur 1 zeigt ein repräsentatives Ergebnis mit AFLIP erhalten. Die Signale in Spur 1 kommen aus dem 125 I-Ligand kovalent an entweder der Zebrabärbling Betaglycan Core - Protein (BG Kern unterhalb der 150 kDa - Marker) verknüpft oder dem BG Kern, der zu seiner Proteoglykan Form verarbeitet wurde , durch Anbringung von Glykosaminoglykanen (GAG, Abstrich im Bereich von 170 kDa an die Spitze des Gels). Dieses Muster der Migration, ein scharfer Core - Protein p...

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Affinitatsmarkierung Nachweis endogener Receptors von Zebrafischembryonen

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Research

JoVE Journal

Developmental Biology

5.6K Aufrufe.  Universidad Nacional Autónoma de México. A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Serie: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Labeling und Imaging Cells in der Zebrafisch Hinterhirn

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Forschung

Neurowissenschaften

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imaging subzellulärer Strukturen in der Living Zebrafischembryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Entwicklungsbiologie

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualisierung von Multiciliated Zellen in der Zebrabärbling durch ein kombiniertes Protokoll der ganze Berg Leuchtstofflampen In Situ Hybridisierung und Immunfluoreszenz

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

<meta charset="utf8"></head><body>Fluoreszierende Proteinmarkierung in Zebrafischlarven mittels CRISPR/Cas9-Knock-in

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Genetik

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

Antikörperaufnahme-Assay zur Verfolgung der Notch/Delta-Endozytose während der asymmetrischen Teilung von radialen Zebrafisch-Gliavorläuferzellen

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Lineage Kennzeichnung von Zebrafisch-Zellen mit Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Biologie

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Semi-Automated Imaging von Gewebespezifische Fluoreszenz-in Zebrafischembryonen

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Wohnung Berge Vorbereitung für Beobachtung und Analyse der Zebrafisch-Embryo Proben von Bunt Ganze Berge<em&gt; In situ</em&gt; Hybridisierung

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Affinitatsmarkierung Nachweis endogener Receptors von Zebrafischembryonen

Zusammenfassung

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Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Antikörperaufnahme-Assay zur Verfolgung der Notch/Delta-Endozytose während der asymmetrischen Teilung von radialen Zebrafisch-Gliavorläuferzellen

Lineage Kennzeichnung von Zebrafisch-Zellen mit Laser Uncagable Fluorescein Dextran

Semi-Automated Imaging von Gewebespezifische Fluoreszenz-in Zebrafischembryonen

Wohnung Berge Vorbereitung für Beobachtung und Analyse der Zebrafisch-Embryo Proben von Bunt Ganze Berge