Name: cryosectioning yeast communities for examining fluorescence patterns
Uploaded: 2012-12-26T13:00:00
Description: Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Vi vil gerne takke Jonathan Cooper Lab på Fred Hutchinson Cancer Research Center for tilladelse til at bruge deres kryostat. Dette arbejde blev støttet af NIH og WM Keck Foundation. BM er en Gordon og Betty Moore stipendiat of Life Science Research Foundation. Vi er taknemmelige for Daniel Gottschling for at dele sine fastsættelse protokol med os.

1. Fastsættelse Gær Fællesskaber <ol><li> Dyrke gær samfund på et membranfilter (fx Millipore MF membranfilter, figur 2A). Denne protokol svarer til en typisk samfund på mindre end 2x10 8 celler på en 6 mm diameter membranfilter. </li><li> Bruge et stykke Whatman 541 filterpapir til at dække bunden af en 1 cm diameter godt (figur 2B). Denne Whatman papir vil blive brugt som en bærer til at overføre fællesskabet i senere faser. Der tilsættes 1 ml 100% me...

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Den procedure, der præsenteres her giver en måde at inspicere den interne struktur i gær samfund. The methanol fastsættelse trin gør gær koloni stift 8 og bevarer integriteten af kolonien, men det medfører også krympning 8, der bør tages i betragtning ved fortolkningen af resultaterne. Vi typisk ser omkring 30% krympning i højden af gærkolonier, sammenlignet med kolonier direkte indlejret i OLT uden fastsættelse af 9. For at undgå svind, en alter...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> 100% Methanol </td><td> JT Baker </td><td> 9070-01 </td><td></td></tr><tr><td> Tissue-Tek OCT-forbindelse </td><td> Andwin Scientific </td><td> 4583 </td><td></td></tr><tr><td> Whatman Grade Nr. 541 Quantitative Filter Paper </td><td> Whatman </td><td> 1541-047 </td><td></td></tr><tr><td> Millipore-MF membranfilter </td><td> Millipore </td><td> HAWP04700 </td><td></td></tr><tr><td> Kryostat </td><td> Leica </td><td> CM 1510 S </td><td></td></tr></tbody></table>

cryosectioning yeast communities for examining fluorescence patterns

Mikrober bor typisk i fællesskaber. Den rumlige organisation af celler i et samfund menes at påvirke overlevelse og funktion i samfundet 1. Optiske sektionering teknikker, herunder konfokal og to-foton mikroskopi, har vist sig nyttig til at observere rumlig organisering af bakterie-og archaeal fællesskaber 2,3. En kombination af konfokal billeddannelse og fysisk inddeling af gærkolonier har afsløret interne organisation af celler 4. Imidlertid har direkte optisk sektionering med konfokal eller to-foton mikroskopi kun været i stand til at nå et par cellelag dybt ind gærkolonier. Denne begrænsning er sandsynligvis på grund af stærk lysspredning fra gærceller 4. Her præsenteres en metode baseret på fastsættelse og cryosectioning at opnå rumlige fordeling af fluorescerende celler i Saccharomyces cerevisiae samfund. Vi anvender methanol som fikseringsmiddel middelat bevare den rumlige fordeling af cellerne. Faste samfund er infiltreret med OCT-forbindelse, frosset og cryosectioned i en kryostat. Fluorescensimagografi af sektionerne afslører den interne organisation af fluorescerende celler i samfundet. Eksempler på gær samfund bestående af stammer, der udtrykker røde og grønne fluorescerende proteiner viser potentialet ved cryosectioning fremgangsmåde til at afsløre den rumlige fordeling af fluorescerende celler og den for genekspression i gærkolonier 2,3. Selvom vores fokus har været på Saccharomyces cerevisiae samfund, kan den samme metode potentielt kan anvendes til at undersøge andre mikrobielle samfund.

Fluorescensbilleder af vertikale tværsnit af gær communities indeholdende rød-og grøn-mærkede konkurrerende populationer 6 er vist i figur 3.. Disse samfund består af to prototrofe stammer af gær og deres konkurrence om delte ressourcer, herunder rum, fører til søjleformet mønstre 7, som vises i deres vertikale tværsnit. Opløsninger ned til en enkelt celle kan løses i tværsnit. Figur 3 sammenligner tværsnit af gentagne samfund opnået med (bund) og u...

Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Vi præsenterer en protokol til frysning og cryosectioning gær samfund til at observere interne mønstre af fluorescerende celler. Metoden bygger på methanol-fixing og OLT-indlejring for at bevare den rumlige fordeling af celler uden at inaktivere fluorescerende proteiner inden for et fællesskab.

Cryosectioning Gær Fællesskaber for Undersøgelse Fluorescens Patterns

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

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