Immunocytochemical study of fresh unfixed erythrocyte membranes by quick-freezing and deep-etching method

速冻深蚀法对新鲜未固定红细胞膜的免疫细胞化学研究

• 批准号: 07670007
• 负责人: OHNO Shinichi
• 金额: $ 1.47万
• 依托单位: Yamanashi Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670007/
• 关键词: fresh unfixed erythrocyte; membrane skeleton; deep-etching; quick-freezing; spectrin; デイ-プエッチング法

A quick-freezing and deep-etching method in combination with erythrocyte spitting was used to examine the cytoplasmic aspect of whole-mount human erythrocyte membranes. The immunostained specimens were positioned on specimen holders and quickly frozen in an isopentane-propane mixture (-193ﾟC) cooled in liquid nitrogen. The erythrocyte membranes on the coverslips were freeze-fractured in liquid nitrogen with a scalpel, transferred in an Eiko FD-3AS machine and deeply etched at 2-6x10^<-7> Torr vacuum at -95ﾟC for 15-30 min. Membrane replicas of the exposed cytoplasmic side of erythrocyte membranes were then prepared by evaporation of platinum at an angle of 24^O and subsequently of carbon at an angle of 90^O. The membrane replicas were routinely treated in household bleach and placed on Formvar-coated copper grids. They were examined in a Hitachi H-8100 electron microscope. Electron micrographs were printed from inverted negative films. Various external forces induced alterations in membrane skeletal organization during the splitting procedure. The initial change was elogation in the peripheral part of the membrane skeleton, examined by immunostaining with a monoclonal antispectrin antibody. Under severe stretching conditions, a linear rearrangement of filamentous components was evident ; these were disposed parallel to the rim of the erythrocyte, while the central part of the concavity exhibited a more compacted structure. These changes resulted in a different distribution of membrane skeletal components between central rigid and peripheral flexible areas in biconcave erythrocytes. It is suggested that the reversible membrane skeletal changes in the flexible areas which resist the external forces are important for maintaining the normal framework of biconcave human erythrocytes.

采用快速冷冻深刻蚀法结合红细胞吐液法对全贴装人红细胞膜细胞质进行了检测。将免疫染色的标本放置在标本夹上，在液氮冷却的异戊烷-丙烷混合物（-193 C）中快速冷冻。将盖子上的红细胞膜用手术刀在液氮中冷冻破裂，在Eiko FD-3AS机器中转移，在-95℃下，在2-6 × 10^<-7> Torr的真空条件下深度蚀刻15-30 min。然后通过以24°角蒸发铂和随后以90°角蒸发碳来制备红细胞膜暴露的细胞质侧的膜复制品。膜复制品在家用漂白剂中进行常规处理，并放置在涂有formvar的铜网格上。在日立H-8100电子显微镜下观察。电子显微照片是从倒置的负片上印出来的。在分裂过程中，各种外力引起了膜骨骼组织的改变。最初的变化是在膜骨架的外周部分，用单克隆抗光谱抗体免疫染色检查。在严重拉伸条件下，丝状成分的线性重排是明显的；它们平行于红细胞的边缘，而凹的中心部分则表现出更紧密的结构。这些变化导致双凹红细胞在中央刚性区和周围柔性区之间膜骨架成分的不同分布。提示在抵抗外力的柔性区，可逆的膜骨架变化对于维持双凹红细胞的正常骨架是重要的。

项目成果

Shinichi Ohno et al.: "Dynamic structure of glomerular capillary loop as revealed by anin vivo cryotechnique" Virchows Archiv. 427. 519-527 (1996)

Shinichi Ohno 等人：“体内冷冻技术揭示的肾小球毛细血管袢的动态结构”Virchows Archiv。

Shinichi Ohno et al.: "Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique." Virchows Archiv. 427. 519-527 (1996)

Shinichi Ohno 等人：“体内冷冻技术揭示的肾小球毛细血管袢的动态结构。”

Nobuo Terada et al.: "Membrane skeleton in avian erythrocytes as revealed by the quick-freezing and deep-etching method" Histol.Histopathol.12 (in press). (1997)

Nobuo Terada 等人：“通过快速冷冻和深蚀刻方法揭示的禽类红细胞膜骨架”Histol.Histopathol.12（印刷中）。

Nobuo Terada et al.: "Three-dimensional ultrastructure of in situ membrane skeletons in human erythrocytes by quick-freezing and deep-etching method" Histology & Histopathology. (in press). (1996)

Nobuo Terada等：“通过快速冷冻和深蚀刻方法观察人红细胞原位膜骨架的三维超微结构”组织学

Shinichi Ohno et al.: "Cell biology of kidney glomerulus" Int.Rev.Cytol.166. 181-230 (1996)

Shinichi Ohno 等人：“肾小球细胞生物学”Int.Rev.Cytol.166。