微波辅助超薄切片快速染色技术的优化与应用

Optimization and application of microwave assisted rapid ultrathin section staining

目的优化基于微波辅助的快速超薄切片染色的关键参数,建立一种快速超薄切片染色方法。方法使用微波组织处理仪以不同功率及染色时间分别用乙酸双氧铀(uranium acetate,UA)和枸橼酸铅(lead citrate,LC)对细胞超薄切片进行染色,通过透射电子显微镜观察并拍照细胞超微结构,评价两种染色剂的最佳染色条件,组合UA和LC最佳微波染色条件,获得最佳UA和LC双染色参数。用腺病毒、肠道病毒71型、疱疹病毒和流感病毒感染的细胞样本验证超薄切片快速染色效果。结果微波组织处理仪优选微波辅助超薄切片染色条件分别为功率200 W、UA染色30 s、LC染色20 s;或者功率300 W、UA染色30 s、LC染色30 s。LC浓度1∶6稀释仍可获得满意的染色效果。参数可推广至家用微波炉应用,最佳染色条件为功率320 W,UA染色30 s、LC染色30 s。结论获得了微波辅助的超薄切片染色最佳参数,可应用于细胞、病毒超薄切片的快速染色。

Objective To optimize key parameters based on microwave assisted rapid staining technique for ultrathin section and establish a rapid ultrathin section preparing method.Methods Ultrathin sections were stained respectively with 1%uranium acetate(UA)and lead citrate(LC)for different duration at various microwave power using microwave tissue processor.Then transmission electron microscope(TEM)photographs of cellular ultrastructure were taken and analyzed.The optimized single staining parameters were decided and combined to investigate the optimal microwave assisted UA and LC double staining conditions.The rapid staining effects of ultrathin sections were verified in different viruses including human adenovirus 5(HAd5),herpes simplex virus(HSV),H1N1 influenza virus,enterovirus 71(EV-A71)human infected cells samples.Results The optimized microwave assisted rapid ultrathin section staining parameters are:UA for 30 s and LC for 20 s at power 200 W or UA for 30 s and LC for 30 s at 300 W using microwave tissue processor.1∶6 dilution of original LC concentration could still work well through microwave assistance.The parameters can be extended and applied to domestic microwave ovens,and the optimized staining parameters are UA for 30 s and LC for 30 s at 320 W.Conclusions The optimized parameters of microwave assisted rapid ultrathin section staining were obtained and can be applied in not only cell samples but also different virus ultrathin sections.