Two-color fluorescence labeling in acrolein-fixed brain tissue.

Area of Science:

• Neuroscience
• Microscopy techniques
• Biochemistry

Background:

• Acrolein is a widely used fixative for electron microscopy due to its preservation of ultrastructure and antigenicity.
• Its use in fluorescence microscopy is limited by concerns of autofluorescence and quenching of fluorescent dyes.

Purpose of the Study:

• To develop a protocol for effective fluorescence labeling in acrolein-fixed rat brain tissue.
• To enable high-quality visualization of fluorescent markers using fluorescence microscopy on acrolein-perfused samples.

Main Methods:

• A protocol involving sodium borohydride pretreatment to reduce autofluorescence.
• Subsequent incubation in ethanol and hydrogen peroxide to enhance fluorescence labeling.
• Application to 40-micrometer sections of acrolein-perfused rat brain.

Main Results:

• Effective removal of autofluorescence from acrolein-fixed tissue sections.
• Enhanced visualization of two fluorescent markers.
• Demonstration of high-quality fluorescence labeling achievable in acrolein-fixed samples.

Conclusions:

• A simple protocol allows for high-quality fluorescence labeling in acrolein-fixed tissues.
• This method overcomes previous limitations associated with acrolein fixation and fluorescence microscopy.
• Adjacent sections can be processed for both electron microscopy and fluorescence labeling, facilitating correlative studies.