High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.
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), GPIba 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 10-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. (J Histochem Cytochem 49:809-819, 2001)
Erlandsen
S L
SL
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA. erlan001@umn.edu
Greet Bittermann
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White
J
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Leith
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Marko
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D015703:Antigens, CD / Q000378:metabolism / Q000648:ultrastructure; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D015815:Cell Adhesion Molecules / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D019276:Glycocalyx / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D039661:Integrin beta3; D008855:Microscopy, Electron, Scanning; D019007:P-Selectin / Q000378:metabolism / Q000648:ultrastructure; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism / Q000648:ultrastructure; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism / Q000648:ultrastructure; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism / Q000648:ultrastructure
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D039661:Integrin beta3; D019007:P-Selectin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins