Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as "reactive-tracers" for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.