Immuno EM Study of Parafibromin Distribution in Cells

为了补充以前在共聚焦光显微镜下的免疫标记研究，我们使用免疫电子显微镜（EM）在更高的空间分辨率下确定人胚胎肾细胞中副纤白蛋白的亚细胞分布。Parafibromin与甲状旁腺功能亢进-颌肿瘤家族性癌综合征，以及甲状旁腺癌和甲状旁腺功能亢进有关。将培养的细胞导入塑料毛细管中，在2000大气压的高压冷冻机上冷冻，以保存细胞的超微结构。冷冻块用戊二醛固定液在丙酮中冷冻取代，包埋在树脂中，切片，并用抗对异纤溴胺的一抗标记。标本与一种与金偶联的二抗反应，并在能量过滤透射电子显微镜（TEM）中进行数字成像。电镜显示，对非纤白蛋白主要集中在细胞核内，在细胞的其他部位也有一些标记。

To complement previous immunolabeling studies in the confocal light microscope, immuno electron microscopy (EM) was performed with the aim of determining the subcellular distribution of the protein parafibromin in human embryonic kidney cells at higher spatial resolution. Parafibromin is implicated in the hyperparathyroidism-jaw tumor familial cancer syndrome, as well as parathyroid cancer and hyperparathyroidism. Cultured cells were introduced into plastic capillary tubes and frozen at 2000 atmospheres in a high-pressure freezing machine to preserve their cellular ultrastructure. The frozen blocks were freeze-substituted in acetone with glutaraldehyde fixative, embedded in resin, sectioned, and labeled with primary antibodies against parafibromin. Specimens were reacted with a secondary antibody conjugated to gold and were imaged digitally in an energy-filtered transmission electron microscope (TEM). Electron micrographs showed that parafibromin was concentrated mainly in the nucleus with some labeling elsewhere in the cell.