When tissues or smears are treated with the usual preparations of fluorescent antibodies, the accompanying non-specific fluorescence found in the preparations (1-19) has imposed limitations upon the use of the fluorescent antibody technique. Dilution, absorption of the antibody preparation with tissue powders or fresh or lyophflized tissues or ion-exchange resins, and masking of sites by treatment with "normal" globulins coupled with fluorescent dyes of contrasdng color, have all been practiced. The need for such devices seems to depend upon the discreteness of the antigens sought witMn the preparations and the relative thicknesses of the latter. With bacteria and certain intracellular viral particles, one may establish localization with relative ease. In contrast, precise localization of soluble antigens that are present in low and variable concentrations, and of some other types of viral particles, is rendered unsure by the accompanying non-specific fluorescence.The present study was undertaken for the purpose of learning something of the reasons for non-specific fluorescence with the aim that this information would aid in the preparation of fluorescent antibody reagents that stain only at sites of specific antigen deposits.Absorption methods using tissue powders were employed only on a limited scale, and the major effort was directed to the coupled globulins themselves.The ultimate criterion for the specificity of fluorescence was based on determining the inhibition of fluorescence by pretreatment with non-fluorescent antibody. Reliable information as to non-specific fluorescence was obtained also by staining tissues where no antigen-antibody reaction was possible.Individual preparations of fluorescent antibody were found to be hetero-
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