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  • Title: Short time bleaching of fluorescein isothiocyanate. A possible parameter for the specific binding of conjugates in immunofluorescence.
    Author: Schauenstein K, Böck G, Wick G.
    Journal: J Histochem Cytochem; 1980 Sep; 28(9):1029-31. PubMed ID: 6774008.
    Abstract:
    The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.
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