Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols

Abstract

The effect of different fixation solutions on the denaturation of membrane-associated antigens in murine lymphoid cells was determined quantitatively using microfluorometric analysis and a radioimmunoassay. Paraformaldehyde and periodate-lysine-paraformaldehyde solutions preserved the antigenicity of cell surface-associated immunoglobulin (S-Ig) antigens when used in concentrations ranging from 0.01 to 4%. However, glutaraldehyde destroyed the antigenicity of S-Ig and Thy 1.2 molecules at concentrations higher than 0.1%. Electron microscopic analysis of the different fixed cell suspensions, after labelling of the cells with a rabbit anti-mouse immunoglobulin-horseradish peroxidase conjugate (RaM-Ig-HRP) showed that prefixation of the sample with 0.1% glutaraldehyde was optimal for immunoelectron microscopical studies, since this concentration preserved both the antigenicity of membrane-associated antigens as well as the ultrastructure of the cells under study. Prolonged fixation periods affected antibody binding. However, S-Ig molecules denatured at a slower rate than Thy 1.2 molecules. A preparation method for the immunoelectron microscopical localization of lymphoid and non-lymphoid cell types in lymphoid organs is reported.