Fujimoto T, Fujimoto K
Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Japan.
J Histochem Cytochem. 1997 Apr;45(4):595-8. doi: 10.1177/002215549704500411.
We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells. Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.
我们描述了一种简单的快速冷冻方法，用于从单层培养细胞中获得质膜的大断裂面。细胞在薄金箔上生长，倒置在薄铜箔上的一层薄明胶上，然后通过在液氮中预冷的两个镀金铜块之间快速挤压进行冷冻。将冷冻的细胞三明治以金面朝上安装在冷冻断裂装置的冷台上，并用冷断裂刀将三明治分开进行断裂。当将该技术应用于汇合的单层细胞时，获得了上层质膜E面和下层质膜P面的大复制品。目前的金属三明治法简单，不需要任何昂贵的设备，并为后续的组织化学操作提供了质膜的大断裂面。
