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- W190790232 abstract "This chapter describes the experimental protocols used for the preparation of samples for high-resolution digital fluorescence microscopy. For high-resolution digital fluorescence microscopy of microtubules (MTs), Cy3-tagged tubulin subunits are injected in living cells. Injected cells are treated with cytochalasin and nocodazole to disrupt actin filaments and MTs, respectively, and then enucleated by centrifugation. After washing out the drugs, images of fluorescently labeled microtubules in cytoplasts are captured with a cooled charge-coupled device (CCD) camera. In cytoplasts lacking the centrosome, decreased MT stability produces an elevated level of free tubulin and imaging of MTs is complicated by unusually high background fluorescence. Therefore, every effort must be made to improve the signal-to-noise ratio. A sensitive cooled CCD camera is used for capturing images. MTs are labeled with a very bright fluorophore to maximize the signal. Porcine brain tubulin subunits are conjugated with Cy3, which is significantly brighter than many other available fluorophores. The chapter describes the protocol used for the preparation of Cy3-tagged porcine brain tubulin subunits." @default.
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- W190790232 date "2001-01-01" @default.
- W190790232 modified "2023-09-29" @default.
- W190790232 title "Digital fluorescence microscopy of cell cytoplasts with and without the centrosome" @default.
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- W190790232 doi "https://doi.org/10.1016/s0091-679x(01)67004-3" @default.
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