High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets:: Detection of P-selectin (CD62P), GPI-IX complex (CD42a/CD42bα,bβ), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging
High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets:: Detection of P-selectin (CD62P), GPI-IX complex (CD42a/CD42bα,bβ), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging
复制标题
DOI：
10.1177/002215540104900702
复制
发表时间：
2001-07-01
影响因子：
3.2
通讯作者：
Marko, M
中科院分区：
文献类型：
--
作者：
Erlandsen, SL;Bittermann, AG;Marko, M
本研究的目的是开发一种模型，用于检测单个细胞粘附分子（CAM）的糖萼的传播人类血小板使用高分辨率冷冻场发射扫描电子显微镜（cryoFESEM）。基于其独特的地形形状，选择人血小板中的三种表面糖蛋白CAM，即P-选择素（CD 62 P）、GPI-IX复合物中的GPIba（CD 42 a/CD 42 B α、B β）和整联蛋白GPIIbIIIa（CD 41/CD 61）。用IO-nm胶体金间接免疫标记人血小板，然后冷冻固定。在从冷冻固定的样品中升华水后，用Pt单向涂覆部分冷冻干燥的血小板，用碳稳定，并使用高分辨率背散射电子成像在透镜内cryoFESEM中进行检查。通过间接免疫金标记检测CAM，并使用软件程序Sterecon中测量的视差差异分析来确定每种类型CAM的长度。我们的研究结果表明，使用高分辨率cryoFESEM识别和检测单个CAMs的糖萼的功效。在生产具有更细粒度的金属涂层方面的进一步进展，以及成像（立体图像的倾斜和角度）的改进，可以提供与糖基化和多聚体CAM复合物的形成相关的形貌的更好的定义。
The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPlba in the GPI-IX complex (CD42a/CD42b alpha ,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with IO-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes.