Name: two-photon-based photoactivation in live zebrafish embryos
Uploaded: 2010-12-24T15:00:00
Description: Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Dank gaat uit naar Genia Brodsky voor figuur graphics, Vyacheslav Kalchenko, Douglas Lutz, en Leonid Roitman voor technisch advies en assistentie met de twee-foton uncaging, Maayan Tahor en Suliman Elsadin voor technische bijstand; Uwe Strahle zo vriendelijk het verstrekken van de neurogenin1 verslaggever lijn en Amos Gutnick voor reacties op dit manuscript. Het onderzoek in de Levkowitz lab is ondersteund door de Duits-Israëlische Foundation (subsidie ​​nummer 183/2007); Israel Science Foundation (subsidie ​​nummer 928/08) en de Harriet &amp; Marcel Dekker Foundation. GL is een taak van de Tauro Career Development Chair in biomedisch onderzoek.

Beschrijven we een protocol van de cel etikettering gebruik van gekooide fluoresceïne, maar ook andere foto-activeerbare kleurstoffen en eiwitten op dezelfde manier worden gebruikt. 1. Injectie van Gekooide Fluoresceïne <ol><li> Bereid 5% stockoplossing (5mg gekooid-fluoresceïne / 100 pi 0,2 M KCl) van dextran-geconjugeerde 4,5-dimethoxy-2-nitrobenzyl (DMNB) gekooide fluoresceïne (10.000 MW dextran, anionische, Invitrogen, moleculaire probes, Carlsbad, Californië , cat. nee. D-3310). ...

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Foto-activeerbare verbindingen zijn moleculen waarvan de functie wordt gemaskeerd totdat ze worden verlicht met een specifieke golflengte (meestal UV), het induceren van een fotochemische reactie die de moleculen omgezet in een biologisch of chemisch actieve toestand. Deze probes geven een zeer krachtige tools in de celbiologie onderzoek, omdat de activering kan nauwkeurig worden tijdelijk en ruimtelijk gecontroleerd door beperking van hun blootstelling aan licht. Het grote voordeel van mult...

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		<th>Catalogue Number</th>
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<td>Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3310</td>
<td>molecular probes</td>
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<td>Agarose for injection trough and coated plates</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9539</td>
<td>&nbsp;</td>
<tr>
<td>Thin Wall Glass Capillaries with filament</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-6</td>
<td>&nbsp;</td>
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<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Microloader tip</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
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<td>Pneumatic picopump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PV820</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>22290-9</td>
<td>&nbsp;</td>
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<td>Low melting point agarose for embryo mounting</td>
<td>&nbsp;</td>
<td>Ultra Pure LMP agarose</td>
<td>16520100</td>
<td>&nbsp;</td>
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<td>Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
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<td>Fast Red</td>
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two-photon-based photoactivation in live zebrafish embryos

Fotoactivatie van target verbindingen in een levend organisme heeft bewezen een waardevolle benadering van de verschillende biologische processen, zoals de embryonale ontwikkeling, cellulaire signalering en volwassen fysiologie te onderzoeken. In dit opzicht is het gebruik van multi-foton microscopie mogelijk kwantitatieve fotoactivering van een bepaalde licht reagerende agent in diepe weefsels op een enkele cel resolutie. Omdat zebravis embryo&#39;s worden optisch transparant, kan hun ontwikkeling worden gevolgd in vivo. Deze eigenschappen maken de zebravis een perfecte modelorganisme voor het regelen van de activiteit van een groot aantal chemische stoffen en de eiwitten door gericht licht. Hier beschrijven we het gebruik van twee-foton microscopie om de activering van chemisch gekooide fluoresceïne, die op zijn beurt laat ons toe om het lot van cellen te volgen in live zebravis embryo&#39;s induceren. We maken gebruik van embryo&#39;s uiten van een live-genetische mijlpaal (GFP), te lokaliseren en precies te richten alle cellen van belang. Deze procedure kan eveneens worden gebruikt voor exacte licht geïnduceerde activatie van eiwitten, hormonen, kleine moleculen en andere gekooide verbindingen.

Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Multifoton microscopie maakt de controle van een lage energetische fotonen met diepe penetratie optische en minder fototoxiciteit. We beschrijven het gebruik van deze technologie voor live cell etikettering in zebravis embryo&#39;s. Dit protocol kan gemakkelijk worden aangepast voor foto-inductie van diverse licht-responsieve moleculen.

Twee-foton-Based Fotoactivatie in de Live zebravis embryo&#39;s

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

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