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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
  • Here is a 2021 Nature protocols paper that cover tips and practical considerations for tissue clearing and imaging

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)

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