Enzymes suitable for the localization of antigens

POase (Section 10.1.1) in EIH is so universal that the techniques described here have been widely known as immunoperoxidase. The popularity of POase is due to the fact that several H-donors fulfill the requirements of suitable substrates, the enzyme is stable, of moderate size, and affordable. 

Some effort has been devoted to obtain even smaller probes, i.e., smaller enzymes conjugated to Fab. An early candidate was cytochrome c (Singer, 1974) which has only 1/3 of the molecular weight of POase, though its diameter is only 30% smaller, but it has the disadvantage of low enzymatic activity. Digestion of cytochrome c (Section 10.3.1) yields the active site (microperoxidase, MPOase) 

APase has also been used for the probes to localize antigens in cells (Mason and Sammons, 1978), in the form of immune complexes of this enzyme (APAAP Section 11.3.2). Naphthol AS phosphate in combination with Fast Red TR gives an intense red reaction product which counterstains well with hematoxylin, whereas in combination with Fast Blue BBN it produces a blue color. 

GOase is becoming more popular due to the low background staining. It can also be used in combination with POase for sequential double staining (Campbell and Bhatnager, 1976). 

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