Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols.
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.
D000818:Animals; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000032:analysis*; D002469:Cell Separation / Q000379:methods; D005404:Fixatives; D007120:Immunochemistry; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D011863:Radioimmunoassay; D011947:Receptors, Antigen, B-Cell / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology
D000954:Antigens, Surface; D005404:Fixatives; D011947:Receptors, Antigen, B-Cell