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01-03-2018 | Methods Paper

Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations

Authors: D. M. Krolewski, V. Kumar, B. Martin, R. Tomer, K. Deisseroth, R. M. Myers, A. F. Schatzberg, F. S. Lee, J. D. Barchas, W. E. Bunney, H. Akil, S. J. Watson Jr.

Published in: Brain Structure and Function | Issue 2/2018

Abstract

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde–acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.

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Title: Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations
Authors: D. M. Krolewski
V. Kumar
B. Martin
R. Tomer
K. Deisseroth
R. M. Myers
A. F. Schatzberg
F. S. Lee
J. D. Barchas
W. E. Bunney
H. Akil
S. J. Watson Jr.
Publication date: 01-03-2018
Publisher: Springer Berlin Heidelberg
Published in: Brain Structure and Function / Issue 2/2018
Print ISSN: 1863-2653
Electronic ISSN: 1863-2661
DOI: https://doi.org/10.1007/s00429-017-1583-z

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Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations

Abstract

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Abstract

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