A Hydrophobic Tissue Clearing Method for a Rat Brain Tissue Sample

Abstract

Source: Kirchner, K.N., et al., A Hydrophobic Tissue Clearing Method for Rat Brain Tissue. J. Vis. Exp. (2020).

This video demonstrates the method of hydrophobic tissue clearing of a rat brain tissue sample. The sample is sequentially dehydrated in methanol, treated with a dichloromethane and methanol mixture to dissolve lipids and pigments, and transferred to dibenzyl ether for complete tissue clearing, enabling detailed imaging without thin sectioning.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Tissue clearing

1. Incubate the sample in a 20% methanol/ 80% deionized water solution for 1 h at room temperature (RT).
2. Incubate the sample in a 40% methanol/ 60% deionized water solution for 1 h at RT.
3. Incubate the sample in a 60% methanol/ 40% deionized water solution for 1 h at RT.
4. Incubate the sample in an 80% methanol/ 20% deionized water solution for 1 h at RT.
5. Incubate the sample in 100% methanol for 1 h at RT.
6. Incubate in fresh 100% methanol overnight at RT.
7. Incubate in 66% dichloromethane (DCM)/ 33% methanol for 3 h, with shaking, at RT.
8. Incubate in dibenzyl ether (DBE) without shaking. Leave the sample in DBE until clear and store in DBE until imaging.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DBE	Sigma-Aldrich	108014-1KG
DCM	Sigma-Aldrich	270997-100mL
Methanol	Sigma-Aldrich	34860-1l-R