Brain Structure and Function

, Volume 223, Issue 2, pp 987–999 | Cite as

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

  • 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. WatsonJr.
Methods Paper


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.


CLARITY Immunofluorescence Imaging Cortex Vasculature 



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.

Compliance with ethical standards

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.

Supplementary material

429_2017_1583_MOESM1_ESM.pdf (1.4 mb)
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)
429_2017_1583_MOESM2_ESM.pdf (461 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)
429_2017_1583_MOESM3_ESM.pdf (96 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)

ESM_4: 3D visualization of PV+ interneurons after surface rendering using Amira. Color coding represents segmented individual neurons immunostained for PV (white) with signal above threshold and assigned automatically in Amira software during quantitation. Video can be viewed using QuickTime or VLC media player. (MPG 409690 KB)

ESM_5: 3D visualization of cortical PV+ interneurons and lectin-stained vasculature. Mouse brains were perfused with 2% acrylamide and cleared with 4% SDS. This movie first shows a series of xy-plane images acquired on a confocal microscope followed by a maximum intensity projection 3D volume rendering of PV cells (red) and vasculature (green) in Imaris software. Video can be viewed using QuickTime or VLC media player. (MP4 170795 KB)

ESM_6: Maximum intensity projected 3D visualization of coronal slice images acquired with a COLM system. Imaris rendering shows neurons immunostained for PV (red) and tyrosine hydroxylase (white) with lectin-stained vasculature (green) in brain perfused with 2% acrylamide and cleared with 4% SDS. Fly through cortical area represents the region of interest considered for quantitation which was kept consistent across samples and groups. TH+ fibers represent nigrostriatal efferents. Video can be viewed using QuickTime or VLC media player. (MP4 166521 KB)

ESM_7: Magnified right hemisphere view of raw images acquired using a COLM system. Dorsal half of the right hemisphere from a coronal brain slice is visualized in a series of xy-plane images recorded in Amira. PV+ neurons and vasculature are in red and green, respectively. Video can be viewed using QuickTime or VLC media player. (MPG 170208 KB)

ESM_8: Surface-rendered post-quantification animation in Amira. Color coded individual PV+ neurons and lectin-stained vasculature (blue) within the quantified region of interest captured with a COLM system. Video can be viewed using QuickTime or VLC media player. (MPG 287748 KB)

ESM_9: Tyrosine hydroxylase (TH) expressing neuronal cell bodies and fibers. A series of xy-plane images show TH positive cell bodies in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Projections from SNc can be seen innervating striatum via nigrostriatal pathway whereas projections from VTA are directed towards cortex. TH positive cell bodies can also be seen around paraventricular nucleus and zona incerta regions in hypothalamus. Movie derived from brain perfused with 2% acrylamide monomer, cleared with 4% SDS and imaged using a COLM with 10x objective. (MPG 65662 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • D. M. Krolewski
    • 1
  • V. Kumar
    • 1
  • B. Martin
    • 1
  • R. Tomer
    • 2
  • K. Deisseroth
    • 3
  • R. M. Myers
    • 4
  • A. F. Schatzberg
    • 5
  • F. S. Lee
    • 6
  • J. D. Barchas
    • 6
  • W. E. Bunney
    • 7
  • H. Akil
    • 1
  • S. J. WatsonJr.
    • 1
  1. 1.Molecular and Behavioral Neuroscience InstituteUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biological SciencesColumbia UniversityNew YorkUSA
  3. 3.Department of BioengineeringStanford UniversityStanfordUSA
  4. 4.HudsonAlpha Institute for BiotechnologyHuntsvilleUSA
  5. 5.Psychiatry and Behavioral ScienceStanford UniversityStanfordUSA
  6. 6.Psychiatry, Weill Cornell Medical CollegeCornell UniversityNew YorkUSA
  7. 7.Department of PsychiatryUniversity of CaliforniaIrvineUSA

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