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

Area of Science:

• Neuroscience
• Biochemistry
• Histology

Background:

• Tyramide signal amplification (TSA) is a sensitive technique for histochemical analysis.
• Multiplex staining using TSA is challenging due to peroxidase (POD) activity quenching.
• Existing methods require overcoming limitations in simultaneous detection of multiple targets.

Purpose of the Study:

• To develop a detailed protocol for multiplex immunofluorescence (IF) staining in mouse brain sections.
• To enable sensitive, simultaneous detection of multiple targets using a fluorescent TSA system.
• To overcome the challenge of peroxidase quenching in multiplex TSA applications.

Main Methods:

• Utilized a fluorescent tyramide-glucose oxidase (FT-GO) system for signal amplification.
• Employed hydrogen peroxide generated by glucose oxidase for covalent deposition of FT.
• Inactivated antibody-conjugated POD using sodium azide to prevent quenching during multiplex labeling.
• Detailed procedures for tissue preparation, triple FT-GO IF staining, and confocal microscopy are described.

Main Results:

• Successfully implemented a multiplex immunofluorescence staining protocol using FT-GO.
• Demonstrated effective covalent deposition of fluorescent tyramide via FT-GO system.
• Achieved sensitive detection of multiple targets in mouse brain sections without POD quenching.

Conclusions:

• The developed FT-GO based fluorescent TSA system provides a robust method for multiplex IF staining.
• This protocol effectively overcomes the limitations of peroxidase quenching in TSA applications.
• Enables sensitive and simultaneous visualization of multiple targets in complex biological tissues like the mouse brain.