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采用标记线粒体的荧光质粒pDsRed2-Mito转染PC12细胞,分别以95%乙醇、甲醇、丙酮和4%多聚甲醛作为固定剂对筛选后的细胞进行固定,最后在激光共聚焦显微镜下观察不同固定剂对荧光质粒荧光强度的影响.结果表明,95%乙醇或甲醇作为固定剂固定细胞后易导致荧光猝灭,而丙酮或4%多聚甲醛固定细胞后,对质粒荧光强度没有显著影响.不同传代次数对pDsRed2-Mito质粒的荧光强度也没有显著影响.因此,在选择pDsRed2-Mito质粒标记线粒体时,不用考虑传代次数的影响,宜选择丙酮或4%多聚甲醛作为固定剂才能最大化的保持质粒的荧光强度.
PC12 cells were transfected with the fluorescent plasmid pDsRed2-Mito labeled with mitochondria and the cells were fixed with 95% ethanol, methanol, acetone and 4% paraformaldehyde as fixative respectively. Finally, the cells were observed under confocal laser scanning microscope Agent on the fluorescence intensity of the fluorescent plasmids.The results showed that fluorescence quenching was easily induced when cells were fixed with 95% ethanol or methanol, while the fixation of cells with acetone or 4% paraformaldehyde had no significant effect on the fluorescence intensity of the plasmids. The number of passages did not significantly affect the fluorescence intensity of pDsRed2-Mito plasmid.Therefore, when selecting the plasmid pDsRed2-Mito for mitochondrial labeling, regardless of the effect of passage number, acetone or 4% paraformaldehyde should be chosen as the immobilizing agent to maximize Maintain the fluorescence intensity of the plasmid.