Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations. Academic Article uri icon

Overview

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.

publication date

  • December 14, 2017

Research

keywords

  • Acrylamide
  • Brain
  • Fluorescent Antibody Technique
  • Lectins

Identity

PubMed Central ID

  • PMC5828880

Scopus Document Identifier

  • 85038107925

Digital Object Identifier (DOI)

  • 10.1007/s00429-015-1020-0

PubMed ID

  • 29243106

Additional Document Info

volume

  • 223

issue

  • 2