Immunohistological Mærkning af mikrotubuli i Sensorisk Neuron dendritter, tracheae, og muskler i Drosophila Larver Krop Wall
Summary
For at forstå, hvordan komplekse celle former, såsom neuronal dendritter, er opnået under udviklingen, er det vigtigt at være i stand til præcist at analysen mikrotubuli organisation. Her beskriver vi en robust immunohistological mærkning metode til at undersøge mikrotubuli organisering af dendritiske arborization neuron sensoriske dendritter, luftrør, muskler og andre<em> Drosophila</em> Larve krop væg væv.
Abstract
For at forstå, hvordan forskelle i komplekse celle figurer er opnået, er det vigtigt præcist at følge mikrotubuli organisation. Det Drosophila Larvetilstand kroppen væggen indeholder flere celletyper, der er modeller til at studere celler og væv morfogenese. For eksempel tracheae bruges til at undersøge rør morfogenese 1, og de dendritiske arborization (DA) sensoriske neuroner i det Drosophila larve er blevet et primært system til belysning af generelle og neuron-klasse-specifikke mekanismer af dendritiske differentiering 2-5 og degeneration 6 .
Formen på dendritceller grene kan variere betydeligt mellem neuron klasser, og endda mellem forskellige grene af en enkelt neuron 7,8. Genetiske undersøgelser i DA neuroner tyder på, at forskellen cytoskeletal organisation kan ligge til grund morfologiske forskelle i dendritiske gren form 4,9-11. Vi leverer en solid immunologisk mærkning metode på enssay in vivo mikrotubuli organisation i DA sensoriske neuron dendritceller Arbor (figur 1, 2, Movie 1). Denne protokol illustrerer dissektion og immunfarvning første instar larve, et stadie, hvor aktiv sensoriske neuron dendritceller udvækst og forgrenet organisation er forekommende 12,13.
I tillæg til farvning sensoriske neuroner, opnår denne metode robuste mærkning af mikrotubuli organisation i musklerne (film 2, 3), luftrør (Figur 3, Movie 3), og andre organer væg væv. Det er værdifuldt for efterforskerne, der ønsker at analysere mikrotubuli organisation in situ i kroppen væggen, når undersøge mekanismer, der styrer væv og celler form.
Protocol
Discussion
For at forstå, hvordan komplekse celle figurer er nået, er det vigtigt at være i stand til præcist at analysen mikrotubuli organisation. Her beskriver vi en robust immunohistological mærkning metode til analyse mikrotubuli organisering af dendritiske arborization neuron sensoriske dendritter. I tillæg til farvning sensoriske neuroner, opnår denne metode robuste immunohistological farvning af luftrøret, muskler og andre organer væg væv.
Vi bruger denne protokol til at undersøge mik…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker Riken til finansiering. P10-Gal4 var en slags gave Alain Vincent (Université Paul Sabatier, Toulouse, Frankrig).
Materials
| Name of the reagent | Company |
Catalogue number |
Comments (optional) |
|---|---|---|---|
| Forceps | Dumont | 11251-20 | |
| Microscissors | FST | 15000-08 | |
| Mouse anti-α-tubulin (Clone: DM1A) | Sigma | T9026 | Dilution 1/1000 |
| Mouse anti-Futsch (Clone: 22C10), supernatant |
Developmental Studies Hybridoma Bank |
22C10 | Dilution 1/1000 |
| Rat anti-CD8 (Clone: 5H10) | Caltag | MCD0800 | Dilution 1/1000 |
| Alexa Fluor 488 anti-mouse IgG | Invitrogen | A-11001 | Dilution 1/500 |
| Cy3 anti-Rat IgG | Jackson Immunoresearch | 712-166-150 | Dilution 1/200 |
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