Encyclopedia of Experiments: Biology
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Нил Красное окрашивание C. elegans: Метод визуализации липидных капель в фиксированных животных
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- При визуализации липидных капель, внутриклеточных органелл хранения нейтральных липидов, начните с добавления моющего раствора, например, содержащего Triton X-100, в образец.
Затем добавьте Нил красный к образцу и защитить его от света, так как краситель светочувствительный. Фиксация позволяет красителю получить доступ к липидным каплям по всему животному.
При воздействии полярных липидов, таких как фосфолипиды, спектр выбросов Нила Реда находится в более высоких красных длинах волн. При воздействии нейтральных липидов, таких как триацилглицеролы и эфиры стерола в нейтральном ядре липидных капель, спектр выбросов Нила Реда смещается на нижние, желто-золотые длины волн.
Поэтому используйте зеленый канал флуоресцентного микроскопа, который будет собирать выбросы желто-золотой длины волны, чтобы визуализировать нильский красный окрашивание липидных капель по всему животному. В примере протокола мы увидим процедуру окрашивания нила Красного и визуализацию липидных капель в фиксированных образцах.
- Для выполнения Нил Красный липидного окрашивания, пластины червей на нематод роста среды, или NGM, посеянные с конца журнала OP50 E. coli, и расти червей при 20 градусах по Цельсию до ранней стадии L4. Вымойте червей с 1 миллилитр раствора PBST и передать червь подвески на 1,5 миллилитров микрофьюдж трубки.
Центрифуга червей в 560 раз тяжести в течение одной минуты, затем удалить супернатант и повторить PBST мыть, пока кишечная палочка очищается от подвески. Далее, к червю гранулы, добавить 100 микролитров 40% изопропанола, или 60% для окрашивания ORO, и инкубировать образец при комнатной температуре в течение трех минут.
- Добавление изопропанола имеет решающее значение для надлежащей проницаемой червей перед окрашивание.
- Центрифуга червей при 560 раз тяжести в течение одной минуты и удалить супернатант, не нарушая червя гранулы.
- Важно свести к минимуму воздействие света во время этапов центрифугации.
- Теперь, находясь в темноте, добавьте 600 микролитров ранее подготовленного рабочего раствора NR к каждому образцу.
После инкубации, центрифуга червей и удалить supernatant. Затем добавить 600 микролитров PBST и инкубировать образцы в темноте в течение 30 минут, чтобы удалить избыток пятна NR. После спиннинг образцов снова, как и раньше, удалить все, кроме примерно 50 микролитров супернатанта.
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