Postreplication labeling of E-leaflet molecules: Membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM

Autor: John E. Rash, Joseph E. Dinchuk, Timothy J. A. Johnson
Rok vydání: 1987
Předmět:
Zdroj: Journal of Electron Microscopy Technique. 7:1-16
ISSN: 1553-0817
0741-0581
DOI: 10.1002/jemt.1060070102
Popis: Conventional freeze-fracture techniques were combined with immunogold labeling and with plastic embedding and sectioning to analyze the distribution of membrane immunoglobulins (mIgs) and their associated intramembrane particles (IMPS) in E-face replicas of murine B-lymphocyte plasma membranes. Immunogold labels were applied to cells after the process of freeze-fracture and replication. Conventional stereoscopic transmission elec- tron microscopic examination of sectioned, labeled replicas (SLRs) revealed that the gold-labeled mIgs were bound to and localized on the outer leaflets of split and replicated membranes. The gold labels were attached to the external determinants of the mIg molecules, which were retained beneath and contig- uous with the replicated E-faces. The mIgs were also localized on the external surface of unreplicated microvilli. In addition, thick sections examined by high- voltage transmission electron microscopy (HVEM) revealed large expanses of replica with well-resolved IMPS. mIgs colocalized with small-diameter (< 60 A) IMPs in E-face replicas of B-lymphocytes whose mIgs were patched by anti- immunoglobulin. Thus, postreplication E-surface labeling of split and repli- cated membranes is a high-resolution technique that is suitable for the study of membrane protein distribution in E-face replicas and contiguous nonrepli- cated tissue.
Databáze: OpenAIRE
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