Enhanced Multiplexing of Immunofluorescence Microscopy Using a Long-Stokes-Shift Fluorophore

Area of Science:

• Biotechnology
• Molecular Biology
• Microscopy

Background:

• Immunofluorescence labeling and microscopy visualize molecular targets using fluorescent probes.
• Spectral overlap of conventional fluorophores limits multiplexing to three antigens.
• Analyzing multiple antigens is crucial for understanding complex biological systems.

Purpose of the Study:

• To describe a method for multiplexed immunofluorescence labeling and imaging of four target antigens.
• To demonstrate the utility of a long-Stokes-shift fluorophore for enhanced spectral separation.
• To provide protocols for four-probe immunofluorescence labeling and imaging.

Main Methods:

• Utilized a long-Stokes-shift fluorophore in combination with three conventional fluorophores.
• Developed protocols for four-probe immunofluorescence labeling.
• Developed protocols for four-probe immunofluorescence imaging.

Main Results:

• Achieved multiplexed imaging of four target antigens in a single sample.
• Demonstrated excellent spectral discrimination between the four probes.
• Confirmed suitability for sensitive analyses using standard imaging hardware.

Conclusions:

• The described method allows for sensitive multiplexed imaging of four antigens.
• The approach offers flexibility in target antigen selection.
• No specialized equipment or reagents are required beyond commercially available labeling reagents.