A protocol for multiplex immunofluorescence staining with a fluorescent tyramide signal amplification system, FT-GO.

Tyramide signal amplification (TSA) is a highly sensitive enzymatic method for histochemical analysis. However, its application to multiplex staining is limited by quenching the catalytic activity of peroxidase (POD). Here, we provide a detailed protocol for multiplex immunofluorescence (IF) staining in mouse brain sections using a fluorescent TSA system, fluorochromized tyramide‑glucose oxidase (FT-GO). FT-GO utilizes hydrogen peroxide produced by oxidation of glucose by glucose oxidase for covalent deposition of FT onto tissue sections. For multiplex labeling with the TSA system, we inactivate antibody-conjugated POD using sodium azide. We describe tissue section preparation, triple FT-GO IF staining and confocal laser scanning microscopy. For complete details on the use and execution of this protocol, please refer to Yamauchi et al. (2022).