Encyclopedia of Experiments: Biology
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Einbetten von erwachsenen Drosophila-Köpfen in Paraffin: Gewebe für die Histologie vorbereiten
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- Um Fliegen zur Paraffineinbettung zu immobilisieren, schieben Sie die anestheisierte Drosophila vorsichtig durch den Spalt eines Fliegenhalsbandes, so dass ihre Köpfe gleichmäßig auf dem Gerät positioniert sind, während sie noch am Rest des Körpers befestigt sind. Zur Gewebefixierung legen Sie die Halsbänder in Carnoys Lösung. Carnoy es ist ein denaturierendes Fixierungsmittel, das aus Ethanol, Chloroform und Essigsäure besteht.
Um die Probe für die Paraffinininfiltration vorzubereiten, dehydrieren Sie das Gewebe, indem Sie den Kragen durch abgestufte Ethanolkonzentrationen übertragen. Übertragen Sie dann den Kragen in Methylbenzoat, ein organisches Lösungsmittel. Als nächstes legen Sie es in ein Methylbenozoat und geschmolzene Paraffinlösung, während sie erhitzt wird, um sicherzustellen, dass das Paraffin geschmolzen bleibt. Fahren Sie mit einer aufwendigen Reihe von Waschschritten mit vorgeheiztem Paraffin fort, um das Methylbenzol zu entfernen.
Als nächstes legen Sie schnell den Kragen in eine Form und gießen geschmolzenes Paraffin, um es vollständig zu bedecken. Sobald das Paraffin gehärtet ist, entfernen Sie den Block aus der Form und verwenden Sie eine Rasierklinge, um überschüssiges Paraffin zu trimmen. Brechen Sie den Kragen ab und verlassen Sie die Reihe der eingebetteten Köpfe im Paraffinblock. Im Beispielprotokoll werden wir das Verfahren für die Analyse neurodegenerativer Phänotypen vorsehen.
- Um zu beginnen, zuerst verwenden Zange, um das Thema fliegen durch den Hals in die Kragen. Richten Sie alle Köpfe in der gleichen Ausrichtung und stellen Sie sicher, dass keine Schäden am Kopf oder Augen. Schließen Sie augenlose Sinus oculis Fliegen an zufälligen bekannten Positionen, so dass die Reihenfolge der Fliegen leicht in den Abschnitten identifiziert werden kann. Zusätzlich, wenn die Fliegenbelastung von Interesse Licht oder weiße Augen hat, Faden rotäugige Fliegen in Intervallen entlang des Streifens, um sicherzustellen, dass die Farbe vorhanden ist.
Notieren Sie die Reihenfolge der Fliegen zusammen mit der Kragennummer, wenn mehr als eine verwendet wird. Sobald ein Halsband fertig ist, legen Sie es in Carnoys Lösung für 3,5 bis 4 Stunden. Führen Sie eine Reihe von Ethanol, Methylbenzoat und Paraffinwäschen durch, um das Fliegengewebe für die Einbettung vorzubereiten.
Gießen Sie geschmolzenes Paraffin vorsichtig über die Kragen und vermeiden Luftblasen und lassen Sie es über Nacht aushärten. Entfernen Sie die Paraffinblöcke mit den Halsbändern aus dem Tablett. Trennen Sie den Paraffinblock mit einer Rasierklinge vom Kragen und brechen Sie den Kragen sanft ab. Die Körper bleiben im Kragen, während die Köpfe im Paraffinblock bleiben.
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