Kinugasa Satoshi, Tojo Akihiro, Sakai Tatsuo, Fujita Toshiro
Division of Nephrology and Endocrinology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Med Mol Morphol. 2010 Dec;43(4):218-25. doi: 10.1007/s00795-010-0500-9. Epub 2011 Jan 26.
To show the three-dimensional distribution of proteins in renal cells, we applied the immunogold scanning electron microscopy method using vibratome slices. Kidney specimens from puromycin aminonucleoside (PAN) nephrotic rats and controls were obtained after intravenous infusion of human serum albumin and fixed in periodate-lysine-paraformaldehyde solution. Vibratome slices were incubated with anti-human albumin antibody and 25-nm gold-labeled secondary antibody. After silver enhancement, the immunogold particles were clearly observed by backscatter electron imaging, whereas they were ambiguous in the secondary electron image. The images showed a higher resolution of the tissues at an acceleration voltage of 5 mV than at 10 mV. Immunogold-labeled albumin was observed in the lumen and endocytotic vesicles of the proximal tubules, and on the podocyte surface in the PAN nephrotic rats, whereas only a few particles were observed in the controls. In conclusion, silver-enhanced immunogold scanning electron microscopy at low acceleration voltages using vibratome sections can be applicable for detecting the intracellular/extracellular localization of molecules in solid tissues. We succeeded in visualizing the enhanced albumin endocytosis of the proximal tubules and the exocytosis of albumin from podocytes in the nephrotic rats.
为了展示蛋白质在肾细胞中的三维分布，我们应用了免疫金扫描电子显微镜方法，使用振动切片机切片。在静脉注射人血清白蛋白后，从嘌呤霉素氨基核苷（PAN）肾病大鼠和对照大鼠获取肾脏标本，并固定于高碘酸盐 - 赖氨酸 - 多聚甲醛溶液中。振动切片机切片与抗人白蛋白抗体和25纳米金标记的二抗一起孵育。银增强后，通过背散射电子成像可清晰观察到免疫金颗粒，而在二次电子图像中则不清晰。图像显示，在5毫伏加速电压下的组织分辨率高于10毫伏时。在PAN肾病大鼠的近端小管腔和内吞小泡以及足细胞表面观察到免疫金标记的白蛋白，而在对照中仅观察到少数颗粒。总之，使用振动切片在低加速电压下进行银增强免疫金扫描电子显微镜可用于检测实体组织中分子的细胞内/细胞外定位。我们成功地可视化了肾病大鼠近端小管增强的白蛋白内吞作用以及足细胞白蛋白的外排作用。
