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Enzyme Immunohistochemical Methods A. INTRODUCTION Since its inception by Coons et al. (1950) immunofluorescence has been used to great advantage in immunodiagnosis for the localization of antigens in situ and also for titration purposes. Titration by immunofluorescence has been replaced almost completely by El A, whereas for localization studies the fluorescein label is increasingly replaced by enzymes. This approach makes it possible to eliminate some of the problems and deficiencies of the immunofluorescence technique.

A disadvantage is the more cumbersome separation. 1. Plastic Solid Phases Current methods, based on noncovalent, adsorption of one of the immunoreactants to the plastic, are simple, and generally give good results. , Hermann et al. (1979) noted a loss of 68% of adsorbed antigen during the test. Desorption may be highly influenced by the serum used (Dobbins Place and Schroeder, 1980). , 1980). The adsorption was shown to be independent of the net change of the protein, but each protein has a different binding constant.

This yields a product which shows the greatest degree of fluorescence at high pH. 0, at a rate of 5 g/liter. The pH is thereby decreasing to 7. 03% hydrogen peroxide is added. 3) is added. The excitation wavelength is 320 nm (emission at 405 nm). The enzyme giving highest detectability with fluorigenic substrates is ßgalactosidase. The hydrolysis of 4-methylumbelliferyl-D-galactopyranoside yields fluorescent 4-methylumbelliferone. 1% BSA is prepared. 34 ml of a 1% solution of 4-methylumbelliferyl-D-galactopyranoside in A^N'-dimethylformamide is added.

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