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Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue
and super-resolution microscopy.
Authors Lee MY, Mao C, Glaser AK, Woodworth MA, Halpern AR, Ali A, Liu JTC, Vaughan JC
Submitted By Submitted Externally on 3/9/2022
Status Published
Journal Nature protocols
Year 2022
Date Published 2/1/2022
Volume : Pages Not Specified : Not Specified
PubMed Reference 35110740
Abstract Fluorescence microscopy is a vital tool in biomedical research but faces
considerable challenges in achieving uniform or bright labeling. For instance,
fluorescent proteins are limited to model organisms, and antibody conjugates can
be inconsistent and difficult to use with thick specimens. To partly address
these challenges, we developed a labeling protocol that can rapidly visualize
many well-contrasted key features and landmarks on biological specimens in both
thin and thick tissues or cultured cells. This approach uses established
reactive fluorophores to label a variety of biological specimens for
cleared-tissue microscopy or expansion super-resolution microscopy and is termed
FLARE (fluorescent labeling of abundant reactive entities). These fluorophores
target chemical groups and reveal their distribution on the specimens;
amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible
amines on proteins, while hydrazide fluorophores target oxidized carbohydrates.
The resulting stains provide signals analogous to traditional general histology
stains such as H&E or periodic acid-Schiff but use fluorescent probes that are
compatible with volumetric imaging. In general, the stains for FLARE are
performed in the order of carbohydrates, amine and DNA, and the incubation time
for the stains varies from 1 h to 1 d depending on the combination of stains and
the type and thickness of the biological specimens. FLARE is powerful, robust
and easy to implement in laboratories that already routinely do fluorescence


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