细胞和组织中两种线粒体标记方法的比较

Comparison Between Different Mitochondrial Staining Methods in Cell and Tissue Samples

目的比较探针和抗线粒体蛋白抗体标记细胞及组织中线粒体的效果,为相关研究选取适合的标记方法提供参考。方法在经体积分数4%多聚甲醛固定的HepG2细胞中,分别使用100 nmol/L MitoTracker Deep Red探针和抗葡萄糖调节蛋白75(Grp75)抗体标记线粒体;在经体积分数4%多聚甲醛固定的HeLa细胞中,分别使用75 nmol/L、100 nmol/L MitoTracker Deep Red探针和抗Grp75抗体标记线粒体;取人肝组织冰冻切片,经4%多聚甲醛固定后,分别使用150 nmol/L MitoTracker Deep Red和抗Grp75抗体标记线粒体。共聚焦显微镜下观察。结果MitoTracker Deep Red探针标记细胞线粒体的效果不如抗Grp75抗体标记清晰,且在HeLa细胞中有非特异性染色,抗体标记可更清楚地反映线粒体的点状和管状分布;在组织中MitoTracker Deep Red探针标记的单个细胞更均匀,标记线粒体的特异性更高,效果优于抗体染色。结论在细胞中使用抗Grp75抗体标记线粒体效果更好,能直观反映线粒体的分裂融合状态;在组织中使用MitoTracker Deep Red染色效果更佳。

Objective To compare the effects of mitochondria staining between specific mitochondrial fluorescent probes and anti-mitochondrial protein antibody in cell and tissue samples.Methods The HepG2 cells fixed by 4%paraformaldehyde were stained with MitoTracker Deep Red(100 nmol/L)or anti-Grp75 antibody(75 nmol/L or 100 nmol/L).The human healthy liver tissue samples fixed by 4%paraformaldehyde were stained with 150 nmol/L MitoTracker Deep Red or anti-Grp75 antibody.The above stained cell and tissue samples were observed using confocal microscopy.Results We found non-specific staining in HeLa cells and obscure mitochondrial image using MitoTracker Deep Red probes,while clear tubular and punctate distribution using anti-Grp75 antibody.In contrast,we observed more specific and better effects of MitoTracker Deep Red probes-stained liver tissue samples as compared to the antibody.Conclusion To visualize mitochondria,the anti-Grp75 antibody staining worked better on cells and the MitoTracker Deep Red probes are more suitable for tissue samples.