A Comparison of Fixatives and Immunohistochemical Technics for Use with Monoclonal Antibodies to Cell Surface Antigens

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Abstract
A variety of fixatives and four immunohistochemical technics were compared for demonstration of binding of monoclonal antibodies to cell surface antigens in tissue sections. Three recently described monoclonal antibodies, directed against the human leukocyte-common antigen (PHM 1) and antigens present on mononuclear phagocytes (PHM 2, PHM 3), were used. Antigen stability to fixation and heat were first assessed by quantitating the resultant effect on binding of the monoclonal antibodies to a macrophage cell line in vitro, using a radiolabeled second antibody. All fixatives affected their binding, but least effect was seen with formaldehyde-based fixatives and ethanol. Sections of formaldehyde- and ethanol-fixed lymph nodes were then labeled with monoclonal antibodies, and four developing methods were compared: indirect immunofluorescence, indirect immunoperoxidase, 3-layer heterologous peroxidase-antiperoxidase (PAP), and a 4-layer PAP technic. The 4-layer PAP proved to be the most sensitive technic since all three antibodies were localized successfully while both indirect technics only detected PHM 1, and the heterologous PAP method was not successful with any monoclonal antibody. The best combination of specific labeling and adequate morphology was seen with periodate-lysine-paraformaldehyde (PLP) fixation and the 4-layer PAP method.