Loss of antibody binding to prefixed cells: fixation parameters for immunocytochemistry

The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2 and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5-1%, whereas glutaraldehyde should be used at concentrations between 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.