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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We would like to thank Drs. Maria Waselus and Aram Parsegian in reviewing the present manuscript. We also greatly appreciate the efforts of Mr. James Stewart and Drs. Qiang Wei and Elaine Hebda-Bauer for assistance with animal care and obtaining animals for preliminary experiments. For technical advice regarding CLARITY procedures we wish to thank Drs. Robert Thompson and Hui Li as well as Mr. Tom Dixon. This work was supported by NIH: R01MH104261, ONR N00014-12-1-0366, Hope for Depression Research Foundation, and Pritzker Neuropsychiatric Research Consortium.
Conflict of interest
For the current study, we report no financial or non-financial conflict of interest.
Human rights and animal participants statements
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
This work was supported by NIH: R01MH104261, ONR N00014-12-1-0366, Hope for Depression Research Foundation, and Pritzker Neuropsychiatric Research Consortium.
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ESM_1: Relative viscosity of 4% acrylamide monomer solution combined with variable bis-acrylamide content. Post-polymerization of solutions containing 0.0375% (A), 0.025% (B), 0.0125% (C), or 0.00625% (D) bis-acrylamide. Note the transition from solid (A, B) to decreased viscosity (C, D). (PDF 1461 KB)
ESM_2: The effect of 4% SDS clearing time on tissue volume. Volume measurements (length x width x thickness) were obtained for 1–4% acrylamide monomer formulas following 37°C SDS clearing times matching both confocal and COLM experiments. Tissue volumes associated with acrylamide concentrations of 1% (n=4/group, 7 and 14 days), 2% (n=4/group, 8 and 14 days), 3% (n=3/group, 10 and 14 days), and 4% (n=3/group, 12 and 14 days) were not significantly different (p>0.09). (PDF 461 KB)
ESM_3: The effect 88% Histodenz refractive index matching solution on tissue volume. Volume measurements (length x width x thickness) were obtained following 14 day SDS clearing, rinsing in boric acid buffer, and overnight incubation in 88% Histodenz. Analysis was performed on Histodenz-equilibrated samples at room temperature to mimic conditions during image acquisition. 3% acrylamide monomer (n=3/group) was significantly expanded vs. 1% (**p<0.005, n=4/group) and 2% (*p<0.034, n=4/group) acrylamide formulas but not compared to that of 4% (p>0.2, n=3/group). (PDF 95 KB)
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Krolewski, D.M., Kumar, V., Martin, B. et al. Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations. Brain Struct Funct 223, 987–999 (2018). https://doi.org/10.1007/s00429-017-1583-z