Clearing methods

Imaging of thick (>50 um) tissue sections is often improved by clearing of the samples - treating them so that the normally opaque sample becomes clear. In 2007, we tested a number of clearing methods in conjunction with Nan Tang. The results are summarized in this white paper. Since then, other clearing methods have been published:

• Passive Clarity (PACT) clearing has been used successfully by a number of UCSF labs.

• The Scale protocol has been published by the Miyawaki lab, which uses 4M urea to render whole mouse embryos essentially transparent.

• A recent paper shows that dehydration with tetrahydrofuran and clearing with dibenzyl ether preserves GFP fluorescence better than ethanol dehydration and clearing with BABB.

• CLARITY uses electrophoresis with SDS to clear very thick brain tissue.

• SeeDB uses fructose and thioglycerol to clear tissue and appears to work about as well as Scale.

• Here is a detailed protocol for fixing and mounting dragonfly tissue in 2,2-TDE.

• 3DISCO uses tissue dehydration in THF and clearing with dibenzyl ether. It clears quickly and is at least somewhat compatible with GFP.

• Citifluor mounting media are supposed to clear well (Confocal Listserv post from Sean Speese, 10/22/2013).

• A 2013 review compares a variety of these clearing methods.

• A 2021 review covering current clearing methods and imaging cleared tissues.

• A 2021 Nature Protocols paper covering tips and practical considerations for tissue clearing and imaging.

Info from users

• A user using CUBIC had trouble with long-term imaging in an open cuvette because the CUBIC crystalized overnight.

Videos for learning about clearing

• Understanding tissue clearing and tissue clearing technologies (Abcam sponsored, there are additional videos in this series)
• Imaging and clearing organoids
• The Basic Principles of Clearing and Imaging Biological Tissues (Logos biosystems sponsored)