Encyclopedia of Experiments: Biology
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Nil rød farvning af C. elegans: En metode til at visualisere Lipid dråber i faste dyr
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- Ved visualisering af lipiddråber begynder de intracellulære opbevaringsorganeller til neutrale lipider med at tilsætte en vaskemiddelopløsning, f.eks.
Derefter tilsættes Nilrød til prøven og beskytter den mod lys, da farvestoffet er lysfølsomt. Fiksering gør det muligt for farvestoffet at få adgang til lipiddråber i hele dyret. Nilrød er et lipofilt farvestof, hvis fluorescensemissionsspektrum afhænger af polariteten i det lokale lipidmiljø.
Når Nile Red udsættes for polære lipider, såsom fosfolipider, er det i de højere, røde bølgelængder. Når nilrøde emissionsspektider udsættes for neutrale lipider, såsom triacylglyceroler og sterolestere i den neutrale kerne af lipiddråber, skifter Nile Reds emissionsspektrum til de nederste, gulgule bølgelængder.
Brug derfor den grønne kanal for et fluorescerende mikroskop, som vil indsamle de gule guld bølgelængdeemissioner, for at visualisere Nilens røde farvning af lipiddråber i hele dyret. I eksempelprotokollen vil vi se en Nil rød farvningsprocedure og billeddannelse af lipiddråber i faste prøver.
- At udføre Nile Red lipid farvning, plade orme på nematode vækst medium, eller NGM, seedet med sen log OP50 E. coli, og vokse orme ved 20 grader Celsius til begyndelsen af L4 fase. Vask orme med 1 milliliter PBST opløsning og overføre ormen suspension til en 1,5 milliliter mikrofuge rør.
Centrifuge ormene ved 560 gange tyngdekraften i et minut, fjern derefter supernatanten og gentag PBST-vasken, indtil E. coli er ryddet fra suspensionen. Dernæst til ormen pellet tilsættes 100 mikroliter på 40% isopropanol eller 60% til ORO-farvning og inkuber prøven ved stuetemperatur i tre minutter.
- Tilsætningen af isopropanol er afgørende for korrekt gennemtrængning af ormene før farvning.
- Centrifuge ormene ved 560 gange tyngdekraften i et minut og fjern supernatanten uden at forstyrre ormepillen.
- Det er vigtigt at minimere lyseksponeringen under centrifugeringstrinnene.
- Nu, mens i mørke, tilsæt 600 mikroliter af tidligere forberedt NR arbejdsopløsning til hver prøve. Invertere rørene tre gange og fuldt ud blande orme og opløsning. Derefter inkubere prøven i mørke ved stuetemperatur i to timer.
Efter inkubation centrifuge ormene og fjern supernatanten. Tilsæt derefter 600 mikroliter PBST og inkubat prøverne i mørke i 30 minutter for at fjerne den overskydende NR-plet. Efter at have drejet prøverne igen som før, skal du fjerne alle undtagen ca. 50 mikroliter supernatant.
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