Name: labeling of single cells in the central nervous system of drosophila melanogaster
Uploaded: 2013-03-04T12:00:00
Description: Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

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Vi har brugt to lidt forskellige varianter af den enkelte neuron mærkningsmetode i vores laboratorier. Forskellene vedrører embryonindsamlingsteam, dechorionisation, devitellinisation og embryo filetering trin. Figur 1 giver en oversigt over de fælles og divergerende trin varianterne. 1. Fremstilling af mikro-kanyler til Embryo Dissektion og Mikropipetter for Dye Injection <ol><li> Glas nåle til embryo dissektion er hentet fra glaskapillarer (1 mm i diameter og 0,1 mm ...

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En væsentlig fordel ved Drosophila som et modelsystem er, at den tillader analyse af udvikling og funktion af niveauet af enkeltceller. Dette er især nyttigt i forbindelse med nervesystemet, hvor de forskellige celletyper er usædvanlig høj og funktionen og morfologi af naboceller kan være helt forskellige. Fremgangsmåden præsenteres her tillader mærkning af individuelle neuroner med et farvestof, som enten kan omdannes til en permanent farvning eller undersøges direkte ved ...

Kendskab til neuronal morfologi på de enkelte celler er en vigtig forudsætning for at forstå neuronal tilslutningsmuligheder og CNS-funktion. Således fra de tidligste dage af neurovidenskab, har forskere forsøgt at udvikle encellede mærkningsteknikker (se 7 for en historisk behandling af dette spørgsmål). Klassiske metoder, såsom Golgi-farvning giver fremragende opløsning af neuronal morfologi, men er ikke egnet, hvis man søger at mærke en bestemt type neuron i en rettet måde, som forekommer farvning på en tilfældig måde. Udviklingen af ​​metoder til farvning enkelte celler ved intracellulær eller juxtacellular injektion af farvestoffer fra en mikroelektrode rettet kravet om særlig mærkning. Anvendelsen af en enkelt neuron farvestof injektion til Drosophila præsenterede en stor udfordring på grund af den lille størrelse af både organismen og dens neuroner. Ikke desto mindre enkelt neuron farvning af embryonisk Drosophila </em&gt; Neuroner blev opnået i midten af 1980&#39;erne i laboratoriet af Corey Goodman 15. Mens metode har været særdeles nyttigt og har givet nogle vigtige indsigter i mekanismer til neuronal udvikling i Drosophila i løbet af de sidste 20-30 år (f.eks 2, 10), har mange arbejdere veget tilbage fra det, hovedsagelig på grund af dets tekniske krav. Tilgængeligheden i de senere år af genetiske teknikker til neuronal mærkning i Drosophila har også bidraget til upopulær enkelt neuron farvestof injektion. GAL4-rettet ekspression af membran-målrettet GFP-konstruktioner kan give fremragende opløsning af neural morfologi 1, 20. Imidlertid har denne fremgangsmåde visse begrænsninger: den ofte uundgåelige ekspression af GFP i flere celler kan tilsløre strukturen af ​​individuelle neuroner og et GAL4 transmissionsledningen er muligvis ikke tilgængelig til at drive ekspression i en bestemt neuron af interesse. Den MARCM (Mosaic Analysis med et Repressible cellemarkør) metode 8 kan tilvejebringe mærkning af væsentlige enhver neuron på det individuelle celleniveau, men ikke med held anvendt i embryoet og tidlig larve på grund af den langsomme omsætning GAL80-proteinet. I betragtning af disse begrænsninger af genetisk mærkning, mener vi, at en enkelt neuron farvestof indsprøjtning i Drosophila foster forbliver en værdifuld teknik og fortjener bredere anvendelse. For at fremme dette mål, vi giver her en detaljeret beskrivelse af metoden. En illustration af sin magt er leveret af vores nylige hensyn til morfologi af det komplette sæt interneuroner i abdominale neuromeres i slutningen af Drosophila foster 14. Denne undersøgelse, som afslørede både morfologisk variation af individuelle neuronale celletyper og principperne for neuromere organisation i centralnervesystemet hos fosteret, ville ikke have været muligt med andre foreliggende mærkningsmetode.

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td></td><td></td><td></td><td> REAGENSER </td></tr><tr><td> DAB </td><td> Sigma-Aldrich </td><td> <a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank">D-5905</a> </td><td> 2-3 mg / ml i 100 mM Tris-HCI, pH 7,4 </td></tr><tr><td> Dil </td><td> Invitrogen / Molecular Probes </td><td> <a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank">D-282</a> </td><td> 1 mg / ml i ethanol </td></tr><tr><td> <a href="http://www.merckmillipore.com/germany/chemicals/formaldehydloesung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWpOEfVhTl" target="_blank">Formaldehyd</a> </td><td> Merck Millipore </td><td></td><td> 7,4% i PBS </td> &lt;/ Tr&gt; <tr><td> Glycerol </td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">Roth</a> </td><td> 3783 </td><td> 70% i PBS </td></tr><tr><td> Heptan Glue </td><td> Beiersdorf AG </td><td> Cello 31-39-30 * </td><td> fortyndet ca. 1-1 med n-heptan </td></tr><tr><td> PBS </td><td></td><td></td><td> 1x </td></tr><tr><td> TRIS </td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">Roth</a> </td><td> 4855 </td><td></td></tr><tr><td> Vectashield </td><td> Vector Laboratories </td><td> <a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank">H1000</a> </td><td></td></tr><tr><td></td><td></td><td></td><td> UDSTYR </td></tr><tr><td> Konfokalt mikroskop </td><td> Leica </td><td> TCS SP2 </td><td> at se og dokumentere labelings i fluorescens </td></tr><tr><td> DC - amplifier </td><td> Dragan Corporation </td><td> Cornerstone ION-100 </td><td> at udføre labelings </td></tr><tr><td> Dækglas </td><td> <a href="http://www.menzel.de/" target="_blank">Menzel</a> </td><td> BB018018A1 </td><td> 18 x 18 mm </td></tr><tr><td> Dækglas </td><td> <a href="http://www.menzel.de/" target="_blank">Menzel</a> </td><td> BB024060A1 </td><td> 24 x 60 mm </td></tr><tr><td> Dissektionsmikroskop </td><td> Leica </td><td> MZ8 </td><td> at forberede og disect embryoner </td></tr><tr><td> Flat Kapillærer </td><td> <a href="http://www.hilgenberg-gmbh.de/" target="_blank">Hilgenberg</a> </td><td></td><td> ydre diameter 1 mm glas tykkelse 0,1 mm </td></tr><tr><td> Injection Kapillærer </td><td> Science Products </td><td> GB 100 TF 8P </td><td></td></tr><tr><td> Mikromanipulator R </td><td> Leica </td><td></td><td> at udføre labelings </td></tr><tr><td> Model P-97 </td><td> Sutter Instruments </td><td></td><td> at trække kapillærer </td></tr><tr><td> Objekt Slides </td><td> <a href="http://www.marienfeld-superior.com/" target="_blank">Marienfeld Superior</a> </td><td> 1000000 </td><td></td></tr><tr><td> Videnskabelig Microscope </td><td> Zeiss </td><td> Axioskop 2 mot </td><td> se labelings efter fotoomdannelse </td></tr><tr><td> Videnskabelig Microscope </td><td> Olympus </td><td> BX50 </td><td> at udføre labelings </td></tr><tr><td> Sony MC3255 Video Camera </td><td> Sony / AVT Horn </td><td> Sony MC3255 </td><td> at registrere labelings efter fotoomdannelse </td></tr></tbody></table> Tabel 1.

labeling of single cells in the central nervous system of drosophila melanogaster

I denne artikel beskriver vi, hvordan du individuelt mærke neuroner i de embryonale CNS af Drosophila melanogaster ved juxtacellular injektion af lipofile fluorescerende membran markør Dil. Denne metode gør det muligt visualisering af neuronal cellemorfologi i stor detalje. Det er muligt at mærke en celle i centralnervesystemet: cellelegemer af mål-neuroner er visualiseret under DIC optik eller ved ekspression af en fluorescerende genetisk markør, såsom GFP. Efter mærkning, kan Dil blive omdannet til en permanent brun plet ved fotoomdannelse at tillade visualisering af cellemorfologi med transmitteret lys og DIC optik. Alternativt kan Dil-mærkede celler observeres direkte med konfokal mikroskopi, hvilket muliggør genetisk indført fluorescerende reporter-proteiner, der skal colocalised. Teknikken kan anvendes i noget dyr uanset genotype, hvilket gør det muligt at analysere mutante fænotyper ved enkeltcelle-opløsning.

4 viser typiske resultater af teknikken, er beskrevet her. 4A viser et eksempel på en Dil fyldt enkelt interneuron der var rent photoconverted. Det pænt viser mængden af ​​detaljer disse præparater tilbud. Når de betragtes under DIC optik den rumlige sammenhæng af det mærkede celle i det ikke-mærkede omgivende væv bliver synlig, fx positionen af cellelegemet i cortex og af fiberen fremspring i neuropile. Figur 4B er et ...

Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster (Video) | JoVE

Vi præsenterer en teknik til mærkning af enkelte neuroner i centralnervesystemet (CNS) af<em&gt; Drosophila</em&gt; Embryoner, som giver analyse af neuronal morfologi ved enten lystransmission eller konfokal mikroskopi.

Mærkning af enkeltceller i centralnervesystemet hos<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

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En skade Paradigm at undersøge centrale nervesystem reparation i<em&gt; Drosophila</em

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