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Brain Struct Funct. 2018 Mar;223(2):987-999. doi: 10.1007/s00429-017-1583-z. Epub 2017 Dec 14.

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

Author information

1
Molecular and Behavioral Neuroscience Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI, 48109, USA. dkrolews@med.umich.edu.
2
Molecular and Behavioral Neuroscience Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI, 48109, USA.
3
Department of Biological Sciences, Columbia University, New York, NY, USA.
4
Department of Bioengineering, Stanford University, Stanford, CA, USA.
5
HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA.
6
Psychiatry and Behavioral Science, Stanford University, Stanford, CA, USA.
7
Psychiatry, Weill Cornell Medical College, Cornell University, New York, NY, USA.
8
Department of Psychiatry, University of California, Irvine, CA, USA.

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.

KEYWORDS:

CLARITY; Cortex; Imaging; Immunofluorescence; Vasculature

PMID:
29243106
PMCID:
PMC5828880
DOI:
10.1007/s00429-017-1583-z
[Indexed for MEDLINE]
Free PMC Article

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