Aims: 

Immunohistochemistry is widely used to detect the expression of proteins, nerve fibers and blood vessels in tissues sections or whole mount embryos. Currently serial section method is widely used to obtain three-dimensional images of nerve fibers and blood vessels. However, this method is time consuming and suboptimal. The advance of confocal and multiphoton microscope allows imaging of 50–100 mm thick tissue sections. However, very thick tissues cannot still be imaged due to scattering of excitation and emitted lights. In this study we used an optical clearing agent to eliminate scattering in thick tissues.

Methods: 

3–4 mm thick mouse brain slices were stained with fluorescent antibodies. They were then optically cleared with benzyl alcohol and benzyl benzoate mixture (BABB) and imaged with confocal microscopes using different objectives.

Results: 

Thick tissues become transparent after optical clearing and can be imaged up to the working distance allowed by the objectives without losing resolution. The whole length of the nerve fibers can be imaged as it courses through from superficial to deep tissues. We noticed that once tissues were cleared confocal microscope could image as deep into the tissue as multiphoton microscope. We also found that fluorophores can maintain its fluorescence for many months when tissue sections and slices were stored in BABB solution.

Conclusion: 

We were able to image millimeter thick tissues with this method. Since scattering is completely eliminated it was no longer necessary to use multiphoton microscope. We found that this method is much better than serial section method to obtain three-dimensional images.