Name: dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord
Uploaded: 2011-12-13T12:00:00
Description: Watch this Scientific Journal Video about DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord at JoVE.com

Forfatterne vil gerne takke Dr. Alistair Garratt (Max Delbrück Center, Berlin) for nyttige kommentarer. Dette arbejde blev støttet af den fælles forskningscenter (SFB665) af det tyske forskningsråd (DFG).

1. Dissektion procedure Bemærk: Eksperimenterende brug af mus bør følge officielt godkendt retningslinjer for pasning og anvendelse af forsøgsdyr. <ol><li> Før tilberedning og sæt din dissektionsmikroskop og læg den kirurgiske instrumenter er nødvendige for dissektion herunder store og små sakse, store tandede pincet, buede pincet og fire sæt Dumont nr. 5 dissekere pincet (hvoraf to har inside-poleret tips) ( for nærmere oplysninger se tabellen af ​​specifikke reage...

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Stereotype projektion mønstre bestående af begge typer af axonal gren dannelse sammen med nem tilberedning i kombination med brug af faste væv til DII mærkning gør embryonale rygmarven med påsat DRG en positiv model for at studere aksonal forgrening. Anvendelsen af ​​meget små mængder af Dii bruge coated glas nåle giver mulighed for - i modsætning til hovedparten mærkning af DRG - visualisering af små grupper af DRG neuroner og dermed analysen af ​​de enkelte axoner og deres forgreninger mønstre. De...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer (valgfri) </td></tr><tr><td> Stereomikroskopet STEMI DRC </td><td> Zeiss </td><td></td><td></td></tr><tr><td> Fosfatbufferet løsning (PBS) </td><td> Biochrom AG </td><td> L182-50 </td><td></td></tr><tr><td> Paraformaldehyd </td><td> Merck </td><td> 8.18715.1000 </td><td></td></tr><tr><td> Standard kirurgiske sakse </td><td> Fin Science Tools </td><td> 14001-13 </td><td></td></tr><tr><td> Tandede standard pincet </td><td> Fin Science Tools </td><td> 11021-14 </td><td></td></tr><tr><td> Ekstra fine iris saks </td><td> Fin Science Tools </td><td> 14088-10 </td><td></td></tr><tr><td> Buet pincet </td><td> Fin Science Tools </td><td> 11003-13 </td><td></td></tr><tr><td> Dumont No.5 fine tips pincet </td><td> Fin Science Tools </td><td> 11254-20 </td><td></td></tr><tr><td> Dumont No.5 højglanspoleret pincet </td><td> Fin Science Tools </td><td> 11252-23 </td><td></td></tr><tr><td> Vännäs-Tübingen foråret saks </td><td> Fin Science Tools </td><td> 15008-08 </td><td></td></tr><tr><td> Filtrerpapir </td><td> Fisher Scientific </td><td> FB59041 </td><td></td></tr><tr><td> Sylgard 184 </td><td> Verdens Precission Instrumenter </td><td> SYLG184 </td><td></td></tr><tr><td> 100-mm petriskåle </td><td> Greiner </td><td> 663102 </td><td></td></tr><tr><td> 12-ml polypropylen rør </td><td> Carl Roth GmbH </td><td> ECO3.1 &lt;/ Td&gt; <td></td></tr><tr><td> 12-godt agarplade </td><td> Becton Dickinson </td><td> 35-3043 </td><td></td></tr><tr><td> Ethanol </td><td> Merck </td><td> 1.00983.2500 </td><td></td></tr><tr><td> Flaming / Brown mikropipette aftrækker P-97 </td><td> Sutter Instrument Co </td><td></td><td></td></tr><tr><td> Borosilikatglas kapillærer </td><td> Harvard Apparatur </td><td> 30-0066 </td><td></td></tr><tr><td> Dii (1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethyl - indocarbocyanine perchlorat) </td><td> Sigma-Aldrich </td><td> 468495 </td><td></td></tr><tr><td> Mikroskopobjektglas SuperFrost Plus </td><td> Carl Roth GmbH </td><td> H867.1 </td><td></td></tr><tr><td> Glas dække glider </td><td> Carl Roth GmbH </td><td> 1870,2 </td><td></td&gt; </tr></tbody></table>

dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord

Her præsenterer vi en teknik til at mærke de baner af små grupper af DRG neuroner i den embryonale rygmarven ved diffusiv farvning hjælp af lipofile sporstof 1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perchlorat (DII) 1 . En sammenligning af axonal veje af vildtype med de mus linjer, hvor generne er muteret tillader test for en funktionel rolle kandidat proteiner i kontrol af axonal forgrening, som er en væsentlig mekanisme i ledninger i nervesystemet. Aksonal forgrening gør det muligt for den enkelte neuron til at forbinde med flere mål, hvilket giver det fysiske grundlag for den parallelle behandling af oplysninger. Forgreninger ved mellemliggende mål regioner axonal vækst kan skelnes fra terminal arborization. Desuden kan forskellige former for axonal gren dannelse klassificeres afhængigt af, om branching resultaterne fra de aktiviteter af væksten kegle (opdeling eller forsinket bhnching) eller fra den spirende af sikkerhedsstillelser fra Axon akslen i en proces, der kaldes interstitiel forgrening 2 (fig. 1). Den centrale fremskrivninger af neuroner fra DRG tilbyder en nyttig eksperimentelle system for at studere begge typer af axonal forgrening: når deres afferente axoner nå dorsale post zone (DREZ) i rygmarven mellem embryonale dage 10 til 13 (E10 - E13) de vise et stereotypt mønster af T-eller Y-formet forgrening. De to resulterende datter axoner herefter fortsætte under rostralt eller caudale retninger, henholdsvis på dorsolaterale margin på ledningen, og først efter en karensperiode soeskende spire fra disse stamceller axoner at trænge igennem den grå stof (interstitiel forgrening) og projekt til relæ neuroner i bestemte bladplader af rygmarven, hvor de yderligere arborize (terminal forgrening) 3. Dii tracings har afsløret vækst kegler på dorsale post zone i rygmarven, der syntes at være i PRocess for at splitte tyder på, at tvedeling er forårsaget af opsplitningen af væksten kegle selv 4 (fig. 2), har dog andre muligheder blevet diskuteret såvel 5. Denne video viser første, hvordan at dissekere rygmarv fra E12.5 mus forlader DRG vedlagt. Efter fiksering af prøveemnet små mængder af Dii anvendes til DRG brug af glas nåle trukket fra kapillarrør. Efter en inkubation, er mærket rygmarven monteres som en omvendt open-bog forberedelse til at analysere de enkelte axoner ved hjælp af fluorescensmikroskopi.

Watch this Scientific Journal Video about DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord at JoVE.com

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Den stereotype fremskrivninger af sensoriske afferenter i gnavere rygmarven tilbyde et lettilgængeligt eksperimentelle system for at studere aksonal forgrener sig gennem sporing af enkelte axoner.

Dii-mærkning af DRG-neuroner til Study aksonal Forgrening i en hel Mount Udarbejdelse af Mouse Embryonale Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

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