Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.