Encyclopedia of Experiments: Biology
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Coloration rouge du Nil de C. elegans: Une méthode pour visualiser les gouttelettes lipidiques chez les animaux fixes
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- Lors de la visualisation des gouttelettes lipidiques, les organites de stockage intracellulaires pour les lipides neutres, commencent par ajouter une solution détergente, comme celle contenant triton X-100, à l’échantillon. Cela perméabilise les cellules du ver. Ensuite, fixer l’échantillon dans la concentration appropriée d’isopropanol, par exemple 40%.
Ensuite, ajoutez le rouge du Nil à l’échantillon et protégez-le de la lumière, puisque le colorant est sensible à la lumière. La fixation permet au colorant d’accéder aux gouttelettes lipidiques dans tout l’animal. Le rouge du Nil est un colorant lipophile dont le spectre d’émission de fluorescence dépend de la polarité de l’environnement lipidique local.
Lorsqu’il est exposé à des lipides polaires, comme les phospholipides, le spectre d’émission du rouge du Nil se trouve dans les longueurs d’onde rouges les plus élevées. Lorsqu’il est exposé à des lipides neutres, comme les triacylglycérols et les esters stérols dans le noyau neutre des gouttelettes lipidiques, le spectre des émissions de Nile Red se déplace vers les longueurs d’onde inférieures en or jaune.
Par conséquent, utilisez le canal vert d’un microscope fluorescent, qui recueillera les émissions de longueur d’onde jaune-or, pour visualiser la coloration rouge du Nil des gouttelettes lipidiques dans tout l’animal. Dans le protocole d’exemple, nous verrons une procédure de coloration rouge du Nil et l’imagerie des gouttelettes lipidiques dans des échantillons fixes.
- Pour effectuer la coloration des lipides rouges du Nil, plaquez les vers sur le milieu de croissance des nématodes, ou MGF, ensemencés avec le journal tardif OP50 E. coli, et cultivez les vers à 20 degrés Celsius jusqu’au stade L4 précoce. Lavez les vers avec 1 millilitre de solution PBST et transférez la suspension du ver à un tube de microfuge de 1,5 milliliter.
Centrifuger les vers à 560 fois la gravité pendant une minute, puis retirer le supernatant et répéter le lavage PBST jusqu’à ce que l’E. coli soit dégagé de la suspension. Ensuite, à la pastille de ver, ajouter 100 microlitres de 40% isopropanol, ou 60% pour la coloration ORO, et incuber l’échantillon à température ambiante pendant trois minutes.
- L’ajout d’isopropanol est crucial pour une perméabilisation adéquate des vers avant la coloration.
- Centrifuger les vers à 560 fois la gravité pendant une minute et enlever le supernatant sans perturber la pastille de ver.
- Il est important de minimiser l’exposition à la lumière pendant les étapes de centrifugation.
- Maintenant, dans l’obscurité, ajouter 600 microlitres de solution de travail NR préparée précédemment à chaque échantillon. Inverser les tubes trois fois et mélanger complètement les vers et la solution. Puis incuber l’échantillon dans l’obscurité à température ambiante pendant deux heures.
Après l’incubation, centrifuger les vers et enlever le supernatant. Puis ajouter 600 microlitres de PBST et incuber les échantillons dans l’obscurité pendant 30 minutes pour enlever l’excès de taches NR. Après avoir fait tourner les échantillons à nouveau comme avant, enlever tous, sauf environ 50 microlitres de supernatant.
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