Takayama I, Terada N, Baba T, Ueda H, Kato Y, Fujii Y, Ohno S
Department of Anatomy, Yamanashi Medical University, Tamaho, Japan.
Histochem Cell Biol. 1999 Dec;112(6):443-5. doi: 10.1007/s004180050426.
A novel "in vivo cryotechnique" with replica immunoelectron microscopy was developed for detecting caveolin localization on replica membranes prepared directly from living smooth muscle cells. After quick-freezing mouse duodenal walls by our "in vivo cryotechnique", the specimens were prepared for freeze-fracture and deep-etch replica membranes. Then they were treated with 5% SDS and 0.5% collagenase to keep some antigens on the replica membranes. The immunogold method could be used to clarify the localization of the caveolin antigen in relation to three-dimensional ultrastructures of living smooth muscle cells. Our new cryotechnique can provide native organization of functional molecules in living cells.
我们开发了一种结合复型免疫电子显微镜的新型“体内冷冻技术”，用于检测小窝蛋白在直接从活的平滑肌细胞制备的复型膜上的定位。通过我们的“体内冷冻技术”对小鼠十二指肠壁进行快速冷冻后，制备用于冷冻断裂和深蚀刻复型膜的标本。然后用5%十二烷基硫酸钠和0.5%胶原酶处理，以保留复型膜上的一些抗原。免疫金法可用于阐明小窝蛋白抗原相对于活平滑肌细胞三维超微结构的定位。我们的新冷冻技术可以提供活细胞中功能分子的天然组织结构。
