Detection of endogenous and immuno-bound peroxidase--the status quo in histochemistry

The discovery of synthetic dyes goes back to 1856 and launched the development of the whole chemical and pharmaceutical industry. In life sciences synthetic dyes represent indispensable tools for the microscopic and macroscopic level. Small dyes have the advantage of their easy adaptability to various measuring equipments. By way of structural modification of the chromophore portion, dye labels can be tailored that they absorb and emit light at desired wavelengths ranging from the UV to the near infrared region of the spectrum. Assisted by the development of light measuring techniques and the commercial availability of highly sensitive equipment, today luminescent labels represent most sensitive detection tools in life sciences and dominate over chromogen based techniques. However, for detection of active sites of peroxidase (PO) so far fluorescent labels have been confined to only a few substrates while a broad variety of well-established chromogenic techniques exist. This review covers fluorescent and chromogenic approaches for the permanent detection of immuno-bound and endogenous PO-activity in fixed cells and tissues. Thereby the tailoring of suitable dye labels is additionally challenged by two demands: (1) The applied dye (or its precursor) must act as enzyme substrate specifically and (2) the enzymatic impact must furnish an insoluble dye product from easy soluble starting materials in a very quick reaction. Hence it is not surprising that among PO-substrates (and enzyme substrates generally), dye conjugates represent only an exception while most of these labels represent reactive dyes or suitable precursors. Chromogenic and fluorescent approaches for the permanent labeling of enzymatic sites are compiled. Furthermore, various area-spanning PO-detection principles are discussed ranging from transmission light (TLM) and fluorescence light (FLM) microscopy (chromogenes, flourochromes, fluorescent chromogenes, chromogenes with nonlinear optical properties) to correlated transmission electron microscopy (TEM; photoconversion of specific chromogenic reaction products, electron opaque and/or osmiophilic chromogenic substrates). Also, approaches for reflectance laser microscopy (RLM), polarization microscopy (PM), and correlative TLM, FLM, and multiphoton fluorescence microscopy (MFM) are discussed.