eBook: Advancing Neuroscience Research - 22

Preserving Endogenous
Fluorescent Protein Signals for
Volumetric Imaging Using the
X-CLARITY Systems and Reagents
for Tissue Clearing
Step-by-step protocol describes how to obtain images of
endogenous FP signals from transparent mouse brains.
Introduction
Tissue clearing techniques have allowed biologists
to acquire high-resolution volumetric images
without the need to reduce samples to thin serial
sections. One major limitation to some techniques
is preserving the signal from endogenous fluorescent
proteins (FPs). The required dehydration step
of solvent-based tissue clearing methods removes
water molecules from tissues, which are required to
preserve FP signal. Although recent solvent-based
techniques 3DISCO and iDISCO have attempted
to address this issue, these methods still can only
maintain FP emission for a few days. Although other
well-known techniques such as CUBIC, Scale,
and PACT preserve FP emission, they are a significant
time investment and are limited to small tissue
samples. The SWITCH technique utilizes strong
fixatives and high temperatures, resulting in a loss
of FP signal.
In principle, the CLARITY method is the most compatible
for FP imaging but having to construct
one's own electrophoresis chamber with a makeshift
cooling system to prevent overheating is a
challenge to most.
The X-CLARITY™ Tissue Clearing Systems
22

eBook: Advancing Neuroscience Research

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