Encyclopedia of Experiments: Biology
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Nijlrode kleuring van C. elegans: een methode om lipidedruppels bij vaste dieren te visualiseren
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- Bij het visualiseren van lipidedruppels beginnen de intracellulaire opslagorganellen voor neutrale lipiden met het toevoegen van een wasmiddeloplossing, zoals een met Triton X-100, aan het monster. Dit zal de cellen van de worm doordringen. Bevestig vervolgens het monster in de juiste concentratie isopropanol, bijvoorbeeld 40%.
Voeg vervolgens Nile Red toe aan het monster en bescherm het tegen licht, omdat de kleurstof lichtgevoelig is. Fixatie geeft de kleurstof toegang tot lipidedruppels in het hele dier. Nile Red is een lipofiele kleurstof waarvan het fluorescentie-emissiespectrum afhangt van de polariteit van de lokale lipideomgeving.
Bij blootstelling aan polaire lipiden, zoals fosfolipiden, bevindt het emissiespectrum van Nile Red zich in de hogere, rode golflengten. Wanneer het wordt blootgesteld aan neutrale lipiden, zoals triacylglycerolen en sterolesters in de neutrale kern van lipidedruppels, verschuift het emissiespectrum van Nile Red naar de lagere, geelgouden golflengten.
Gebruik daarom het groene kanaal van een fluorescerende microscoop, die de geelgouden golflengte-emissies zal verzamelen, om de Nijlrode kleuring van lipidedruppels in het hele dier te visualiseren. In het voorbeeldprotocol zien we een Nijlrood-kleuringsprocedure en beeldvorming van lipidedruppels in vaste monsters.
- Om Nijlrode lipidenkleuring uit te voeren, plaatwormen op nematodengroeimedium, of NGM, gezaaid met laat log OP50 E. coli, en laat de wormen groeien op 20 graden Celsius tot vroeg L4-stadium. Was de wormen met 1 milliliter PBST-oplossing en breng de wormsuspensie over naar een microfugebuis van 1,5 milliliter.
Centrifugeer de wormen gedurende één minuut op 560 keer de zwaartekracht, verwijder vervolgens het supernatant en herhaal de PBST-wasbeurt totdat de E. coli uit de suspensie is verwijderd. Voeg vervolgens aan de wormkorrel 100 microliters van 40% isopropanol of 60% voor ORO-kleuring toe en incubeer het monster gedurende drie minuten op kamertemperatuur.
- De toevoeging van isopropanol is cruciaal voor een goede permeabilisatie van de wormen vóór het kleuren.
- Centrifugeer de wormen gedurende één minuut op 560 keer de zwaartekracht en verwijder het supernatant zonder de wormkorrel te verstoren.
- Het is belangrijk om de blootstelling aan licht tijdens de centrifugeerstappen te minimaliseren.
- Voeg nu, terwijl u in het donker bent, 600 microliter eerder bereide NR-werkoplossing toe aan elk monster. Keer de buizen drie keer om en meng de wormen en de oplossing volledig. Incubeer het monster vervolgens twee uur in het donker bij kamertemperatuur.
Na incubatie centrifugeer je de wormen en verwijder je het supernatant. Voeg vervolgens 600 microliter PBST toe en incubeer de monsters gedurende 30 minuten in het donker om de overtollige NR-vlek te verwijderen. Verwijder na het draaien van de monsters opnieuw zoals voorheen alle behalve ongeveer 50 microliter supernatant.
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