Quantitative immunohistochemistry

In general there exist two possibilities to obtain quantitatively information in immunohistochemical work: (1) Titration of an antibody of defined specificity and concentration on different antigenic substrates. (2) The measurement of the "specific signal" of the marker molecule used for the visualization of the specific fixation of antibody to the homologous antigen in histological preparations. Examples for both kinds of approach are given by recent data from quantitative immunofluorescence (IF) studies performed in this laboratory. In the second part of this talk some theoretical aspects of quantitative IF are discussed concerning the fluorescence properties of fluorescein isothiocyanate, the most widely used fluorochrome. Macrospectrofluorometric measurements revealed marked effects on the emission intensity of the composition of the embedding medium and the concentration of the dye. Furthermore, the conjugation to protein is shown to cause a considerable quenching of UV excited fluorescence, but not at excitation with visible blue (496 nm). A further critical point in quantitative IF is the illumination source. The great advantages of laser light excitation as compared to filtered light of the most widely used mercury arcs are discussed. Finally some experimental data concerning the recovery of already faded fluorescence as observed with short, repeated laser light pulses are mentioned. The practical significance of these theoretical data are stressed.