Encyclopedia of Experiments: Biology
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Embedding Voksen Drosophila Hoder i Paraffin: Forbereder vev for histologi
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- For å immobilisere fluer for parafininnbygging, skyv forsiktig bedøvet Drosophila gjennom gapet på en fluekrage slik at hodene deres er jevnt plassert på toppen av enheten mens de fortsatt er festet til resten av kroppen. For vevsfiksering, plasser kragene i Carnoys løsning. Carnoy's er et denatureringsfikseringsmiddel som består av etanol, kloroform og eddiksyre.
For å forberede prøven for parafininfiltrasjon, dehydrerer du vevet ved å overføre kragen gjennom graderte konsentrasjoner av etanol. Deretter overfører du kragen til metylbenzoat, et organisk løsningsmiddel. Deretter plasserer du det i en metylmonozoat og smeltet parafinløsning mens den oppvarmes for å sikre at parafinen forblir smeltet. Fortsett til en forseggjort serie vasketrinn med forvarmet parafin alene for å fjerne metylbenzoatet.
Deretter plasserer du kragen raskt i en mugg og hell smeltet paraffin for å dekke den helt. Når parafinen er herdet, fjern blokken fra formen og bruk et barberblad for å trimme overflødig paraffin. Bryt av kragen og la raden med innebygde hoder i parafinblokken. I eksempelprotokollen vil vi se prosedyren demonstrert for analyse av nevrodegenerative fenotyper.
- For å begynne med, bruk først tang for å tre motivet flyr i nakken inn i kragene. Juster alle hodene i samme retning og sørg for at det ikke oppstår skade på hodet eller øynene. Inkluder øyeløse sinusokulisfluer i tilfeldige kjente posisjoner, slik at fluens rekkefølge lett kan identifiseres i avsnittene. I tillegg, hvis flystammen av interesse har lyse eller hvite øyne, tråd rødøyde fluer i intervaller langs stripen for å sikre tilstrekkelig pigment er til stede for å fargelegge flekken.
Registrer rekkefølgen på fluene sammen med kragenummeret hvis mer enn en brukes. Når en krage er ferdig, plasser den i Carnoys løsning i 3,5 til 4 timer. Utfør deretter kragene i en gummiisbitbrett en per seksjon.
Hell forsiktig smeltet parafin over kragene og unngå luftbobler og la den herdes over natten. Fjern parafinblokkene som inneholder kragene fra brettet. Skill parafinblokken fra kragen med et barberblad, og brekk forsiktig av kragen. Kroppene forblir i kragen mens hodene holder seg i parafinblokken.
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