Looking through Brains with Fast Passive CLARITY: Zebrafish, Rodents, Non-human Primates and Humans

Farzad Mortazavi; Alexander J Stankiewicz; Irina V Zhdanova

doi:10.21769/BioProtoc.3321

Looking through Brains with Fast Passive CLARITY: Zebrafish, Rodents, Non-human Primates and Humans

Bio Protoc. 2019 Aug 5;9(15):e3321. doi: 10.21769/BioProtoc.3321.

Authors

Farzad Mortazavi¹, Alexander J Stankiewicz^{2

3}, Irina V Zhdanova³

Affiliations

¹ Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, MA 02118, USA.
² Department of Physics, University of Connecticut, Storrs, Connecticut 06269, USA.
³ Department of Preclinical Research, BioChron LLC, Worcester, Massachusetts 01605, USA.

Abstract

Recently developed CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tis-sue-hYdrogel) technique renders the tissue transparent by removing lipids in the tissue, while preserving and stabilizing the cellular and subcellular structures. This provides effective penetration of diverse labeling probes, from primary and secondary antibodies to complementary DNA and RNA strands. Followed by high-resolution 3D imaging of neuronal cells and their projections in thick sections, tissue blocks, whole brains, or whole animals, CLARITY allows for superior quantitative analysis of neuronal tissue. Here, we provide our detailed protocol for PACT (Passive Clarity Technique) in brain tissue of diverse species, including human, non-human primate, rodents, and zebrafish. We describe the six principal steps: (1) Tissue fixation and preparation, (2) Passive lipid removal, (3) Immuno-labeling, (4) Optical clearing, (5) Imaging, (6) 3D visualization and quantification.

Keywords: Adult neurogenesis; Brain; CLARITY; Human; Imaging; Immunohistochemistry; Mice; Non-human primate; Rat; Rhesus monkey; Whole-tissue staining; Zebrafish.