Fujita Akikazu, Fujimoto Toyoshi
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa, Nagoya, 466-8550, Japan.
Histochem Cell Biol. 2007 Nov;128(5):385-9. doi: 10.1007/s00418-007-0341-3. Epub 2007 Sep 18.
SDS-digested freeze-fracture replicas have been used as a substrate for immunoelectron microscopy to determine localization of membrane proteins and lipids. We, as well as others, have noticed that replicas prepared by first evaporating carbon are labeled more efficiently than conventional preparations in which platinum/carbon is evaporated first followed by carbon. In the present study, we examined whether the superior labeling in the carbon-first replica is caused by better retention of membrane molecules during SDS digestion. We used phosphatidylcholine liposomes as a model sample and measured the amount of inorganic phosphorus retained in the SDS-digested replica. The result showed that there was equivalent retention of inorganic phosphate among replicas prepared in different ways, indicating that labeling intensity on the replica did not correlate with the retention ratio. Interestingly, despite a similar retention ratio, replicas made of carbon alone gave far less labeling for ganglioside GM1 and phosphatidylcholine than replicas prepared by carbon followed by platinum/carbon. These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling.
十二烷基硫酸钠（SDS）消化的冷冻蚀刻复制品已被用作免疫电子显微镜的底物，以确定膜蛋白和脂质的定位。我们以及其他人都注意到，先蒸发碳制备的复制品比先蒸发铂/碳再蒸发碳的传统制备方法标记效率更高。在本研究中，我们研究了碳先复制品中更好的标记是否是由于在SDS消化过程中膜分子保留得更好。我们使用磷脂酰胆碱脂质体作为模型样品，并测量了SDS消化复制品中保留的无机磷含量。结果表明，不同制备方法的复制品中无机磷酸盐的保留量相当，这表明复制品上的标记强度与保留率无关。有趣的是，尽管保留率相似，但仅由碳制成的复制品对神经节苷脂GM1和磷脂酰胆碱的标记远少于由碳然后铂/碳制备的复制品。这些结果表明，探针与被碳捕获的脂质结合比与被铂捕获的脂质结合更有效。尽管如此，在碳之后蒸发铂对于正确标记是必不可少的。
