Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue

Area of Science:

• Neuroscience
• Histology
• Microscopy

Background:

• Lipofuscin, an age-related fluorescent pigment, accumulates in the central nervous system (CNS) cytoplasm.
• Lipofuscin's broad excitation/emission spectra interfere with fluorescence microscopy techniques like retrograde tract tracing and immunocytochemistry.
• Existing methods lack effective strategies to reduce lipofuscin autofluorescence without compromising specific fluorescent signals.

Purpose of the Study:

• To evaluate chemical treatments for reducing lipofuscin-like autofluorescence in neural tissue sections.
• To assess the impact of these treatments on other fluorescent labels used in microscopy.
• To identify optimal methods for minimizing autofluorescence while preserving specific signals.

Main Methods:

• Tested chemical treatments, including copper sulfate (CuSO4) and Sudan Black B (SB), on neural tissue sections from various species (monkey, human, rat).
• Quantified the reduction in lipofuscin autofluorescence.
• Evaluated the effect of treatments on the intensity of immunofluorescent labels and fluorescent retrograde tracers.

Main Results:

• 1-10 mM CuSO4 in ammonium acetate buffer (pH 5) and 1% SB in 70% ethanol significantly reduced or eliminated lipofuscin autofluorescence.
• These treatments caused only a slight reduction in the intensity of specific fluorescent labels (immunofluorescence and retrograde tracers).
• The reduction in specific fluorophores was considerably less pronounced than the reduction in lipofuscin autofluorescence.

Conclusions:

• CuSO4 and SB treatments offer a viable solution for reducing lipofuscin-induced autofluorescence in neural tissue.
• These methods provide a practical compromise, effectively minimizing autofluorescence while maintaining the integrity of specific fluorescent labels.
• The findings support the use of CuSO4 or SB for enhanced clarity in fluorescence microscopy of aged neural tissues.