Encyclopedia of Experiments: Biology
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Intégrer des têtes adultes de drosophile dans la paraffine : Préparer les tissus à l’histologie
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- Pour immobiliser les mouches pour l’intégration de paraffine, poussez soigneusement la drosophile anesthésiée à travers l’espace d’un collier volant afin que leurs têtes soient uniformément placées sur le dessus de l’appareil tout en restant attachées au reste du corps. Pour la fixation des tissus, placez les colliers dans la solution de Carnoy. Carnoy est un agent de fixation dénaturant composé d’éthanol, de chloroforme et d’acide acétique.
Pour préparer l’échantillon à l’infiltration de paraffine, déshydrater le tissu en transférant le collier à travers des concentrations classées d’éthanol. Ensuite, transférez le collier au benzoate méthylique, un solvant organique. Ensuite, placez-le dans un benozoate méthylique et une solution de paraffine fondue pendant qu’il est chauffé pour s’assurer que la paraffine reste fondue. Passez à une série élaborée d’étapes de lavage avec de la paraffine préchauffée seule pour enlever le benzoate méthyle.
Ensuite, placez rapidement le collier dans un moule et versez de la paraffine fondue pour le couvrir complètement. Une fois que la paraffine s’est durcie, retirez le bloc du moule et utilisez une lame de rasoir pour couper l’excès de paraffine. Détachez le collier en laissant la rangée de têtes intégrées dans le bloc de paraffine. Dans le protocole d’exemple, nous verrons la procédure démontrée pour l’analyse des phénotypes neurodégénératifs.
- Pour commencer, tout d’abord, utiliser des forceps pour enfiler les mouches du sujet par le cou dans les colliers. Alignez toutes les têtes dans la même orientation et assurez-vous qu’il n’y a pas de dommages à la tête ou aux yeux. Inclure des mouches sinusoïstes sans yeux à des positions connues aléatoires afin que l’ordre des mouches puisse être facilement identifié dans les sections. En outre, si la souche d’intérêt de la mouche a les yeux clairs ou blancs, enfiler les mouches à yeux rouges à intervalles le long de la bande pour s’assurer qu’un pigment suffisant est présent pour tacher la toboggan.
Enregistrez l’ordre des mouches avec le numéro du collier si plus d’un collier est utilisé. Une fois qu’un collier est terminé, placez-le dans la solution de Carnoy pendant 3,5 à 4 heures. Effectuez une série de lavages à l’éthanol, au benzoate méthylique et à la paraffine pour préparer le tissu volant à l’intégration. Ensuite, placez les colliers dans un bac à glaçons en caoutchouc un par section.
Verser délicatement la paraffine fondue sur les cols en évitant les bulles d’air et laisser durcir pendant la nuit. Retirer les blocs de paraffine contenant les colliers du plateau. Séparer le bloc de paraffine du col à l’aide d’une lame de rasoir, en cassant doucement le collier. Les corps resteront dans le col pendant que les têtes resteront dans le bloc de paraffine.
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