A Hydrophobic Tissue Clearing Method for a Rat Brain Tissue Sample

Begin with a brain tissue sample, which is non-transparent due to the presence of pigments and lipids. These components cause light scattering in fluorescently labeled neurons.

Sequentially incubate the tissue in solutions with increasing methanol concentrations to remove water from it and dehydrate the tissue gradually.

Then, treat this tissue with concentrated methanol to eliminate residual water, ensuring complete dehydration. 

Next, transfer the tissue to an initial clearing solvent containing dichloromethane and methanol. 

Incubate with gentle shaking to allow the solvent to penetrate the tissue deeply.

Dichloromethane removes pigments and lipids from tissue, reducing light scattering and enhancing transparency.

Finally, transfer the tissue to dibenzyl ether, a hydrophobic solvent and incubate without shaking. 

The solvent acts as the final clearing agent that further enhances tissue transparency.  

This process improves light penetration, enabling clearer visualization of labeled neurons and their connections in the brain tissue sample.