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THE TWO-DIMENSIONAL TOPOGRAPHIC DISTRIBUTION OF H-2 HISTOCOMPATIBILITY ALLOANTIGENS ON MOUSE RED BLOOD CELL MEMBRANES

Authors
Journal
The Journal of Cell Biology
0021-9525
Publisher
The Rockefeller University Press
Publication Date
Keywords
  • Brief Notes
  • Article
Disciplines
  • Biology

Abstract

THE TWO-DIMENSIONAL TOPOGRAPHIC DISTRIBUTION OF H-2 HISTOCOMPATIBILITY ALLOANTIGENS ON MOUSE RED BLOOD CELL MEMBRANES INTRODUCTION In connection with studies of the molecular organization of biological membranes (13) we have been interested in determining the two-dimen- sional topographical distribution of specific components on membranes. Electron microscopy with ferritin-antibody (12) and ferritin-plant agglutinin (9) conjugates can be used to localize specific antigens and oligosaccharide components, respectively, with a resolution of about 300 A . Labeled-antibody techniques have previously been used to study specific surface antigens of various cell types (cf. Refs. 1, 4-7) . In earlier studies, however, the labeled cells had been embedded and sectioned before examination in the electron microscope; in such electron micrographs, es- sentially only the one-dimensional distribution of an antigenic component is revealed . In order to determine the two-dimensional surface distribu- tion, we have developed an alternative method of specimen preparation (9) . Cells are lysed at an air-water interface, which causes the entire cell membrane to spread out flat at the interface . The flattened-out membranes are then picked up on an electron microscope grid, the ferritin-antibody stain is applied to the membrane on the grid, and the grid is then examined by direct transmission electron microscopy . In this note, we demonstrate the use of this technique in conjunction with the indirect ferritin- antibody staining method to visualize the two- dimensional distribution of the H-2 histocom- patibility alloantigens (2) over large areas of the mouse red blood cell membrane . MATERIALS AND METHODS Reagents and Antisera Horse spleen ferritin (6 X recrystallized, Miles Labs, Inc., Kankakee, Ill.) was further purified by cadmium sulfate crystallization, ammonium sulfate precipitation, and ultracentrifugation (8) . Rabbit GARTH

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