Encyclopedia of Experiments: Biology
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Colorazione rossa del Nilo di C. elegans: Un metodo per visualizzare le goccioline lipidiche negli animali fissi
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- Quando si visualizzano le goccioline lipidiche, gli organelli di stoccaggio intracellulari per lipidi neutri, iniziano aggiungendo una soluzione detergente, come una contenente Tritone X-100, al campione. Ciò permeabilizzerà le cellule del verme. Successivamente, fissare il campione nella concentrazione appropriata di isopropanolo, ad esempio il 40%.
Quindi, aggiungere Nile Red al campione e proteggerlo dalla luce, poiché il colorante è sensibile alla luce. La fissazione consente al colorante di accedere alle goccioline lipidiche in tutto l'animale. Nile Red è un colorante lipofilo il cui spettro di emissione di fluorescenza dipende dalla polarità dell'ambiente lipidico locale.
Pertanto, utilizzare il canale verde di un microscopio fluorescente, che raccoglierà le emissioni di lunghezza d'onda giallo-oro, per visualizzare la colorazione rosso del Nilo delle goccioline lipidiche in tutto l'animale. Nel protocollo di esempio, vedremo una procedura di colorazione rosso del Nilo e l'imaging di goccioline lipidiche in campioni fissi.
- Per effettuare la colorazione lipidica del Nilo Red, i vermi piastra sul mezzo di crescita del nematode o NGM, seminati con log tardivo OP50 E. coli, e far crescere i vermi a 20 gradi Celsius fino all'inizio dello stadio L4. Lavare i vermi con 1 millilitro di soluzione PBST e trasferire la sospensione del verme in un tubo di microfugo da 1,5 millilitri.
Centrifugare i vermi a 560 volte la gravità per un minuto, quindi rimuovere il supernatante e ripetere il lavaggio PBST fino a quando lE. coli non viene eliminato dalla sospensione. Successivamente, al pellet di verme, aggiungere 100 microlitri del 40% di isopropanolo, o il 60% per la colorazione ORO, e incubare il campione a temperatura ambiente per tre minuti.
- L'aggiunta di isopropanolo è fondamentale per una corretta permeabilizzazione dei vermi prima della colorazione.
- Centrifugare i vermi a 560 volte la gravità per un minuto e rimuovere il supernatante senza interrompere il pellet di verme.
- È importante ridurre al minimo l'esposizione alla luce durante le fasi di centrifugazione.
- Ora, mentre si è al buio, aggiungere 600 microlitri di soluzione di lavoro NR precedentemente preparata a ciascun campione. Invertire i tubi tre volte e mescolare completamente i vermi e la soluzione. Quindi incubare il campione al buio a temperatura ambiente per due ore.
Dopo l'incubazione, centrifugare i vermi e rimuovere il supernatante. Quindi aggiungere 600 microlitri di PBST e incubare i campioni al buio per 30 minuti per rimuovere la macchia NR in eccesso. Dopo aver ruotato nuovamente i campioni come prima, rimuovere tutti tranne circa 50 microlitri di supernatante.
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