Encyclopedia of Experiments: Biology
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Transcript
Please note that all translations are automatically generated.
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-脂質滴を可視化する場合、中性脂質用の細胞内貯蔵小器官は、トリトンX-100を含むような洗剤溶液をサンプルに加えることで始まる。
次に、試料にナイルレッドを加え、色素が光に敏感であるため、色素を光から保護します。固定は、色素が動物全体の脂質滴にアクセスすることを可能にします。
リン脂質などの極性脂質に曝露されると、ナイルレッドの発光スペクトルは高い赤色波長である。
したがって、黄色金波長の放射を収集する蛍光顕微鏡の緑色チャネルを使用して、動物全体の脂質滴のナイルレッド染色を視覚化します。
- ナイルレッド脂質染色、線虫増殖培地上のプレートワーム、またはNGMを遅ログOP50大腸菌で播種し、20°Cから早期L4段階でワームを増殖させる。
ワームを重力560倍で1分間遠心分離し、上清を取り除き、大腸菌が懸濁液から取り除くまでPBST洗浄を繰り返します。
- イソプロパノールの添加は、染色前にワームの適切な透過性のために重要です。
- 1分間560倍の重力でワームを遠心分離し、ワームペレットを破壊することなく上清を除去します。
-遠心分離工程での光の露出を最小限に抑えることが重要です。
- 今、暗闇の中で、各サンプルに以前に準備されたNR作業溶液の600マイクロリットルを追加します。チューブを3回反転し、ワームと溶液を完全に混ぜます。
インキュベーションに続いて、ワームを遠心分離し、上清を取り除く。その後、PBSTの600マイクロリットルを追加し、余分なNR染色を除去するために30分間暗闇の中でサンプルをインキュベートします。
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