Journal Article

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

Greenbaum, Alon, Gradinaru, Viviana, Lignell, Antti, Deverman, Benjamin E, Flytzanis, Nicholas C, Bjorkman, Pamela J, Fowlkes, Charless C, Chan, Ken Y, Yang, Bin, Ladinsky, Mark S, Xiao, Cheng, Treweek, Jennifer B, Cai, Long

2015

Overview

This protocol describes how to fix, embed, clear and stain excised organs or whole organisms to create optically transparent samples. This versatile protocol is able to process a wide range of sample types for high-resolution imaging. To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/114/1564 / 631/1647/245/2221 / 631/378/2650 / 631/80/2373 / Analysis / Analytical Chemistry / Animals / Biological research / Biological specimens / Biological Techniques / Biology, Experimental / Biomolecules / Caenorhabditis elegans / Cellular communication / Cellular signal transduction / Circuits / Clearing / Computational Biology/Bioinformatics / Datasets / Detergents - isolation & purification / Embedding / Extraction (Chemistry) / Fluorescence / Fluorescence microscopy / Genetic aspects / High resolution / Histocytochemistry - methods / Hybrids / Hydrogels / Image resolution / Life Sciences / Light scattering / Lipids / Lipids - isolation & purification / Methods / Mice / Microarrays / Optical Imaging - methods / Organic Chemistry / Organisms / Organs / Pathology - methods / Perfusion / Permeability / Phenotype / Phenotyping / Physiological aspects / Protocol / Rats / Refractivity / Specimen Handling - methods / Staining and Labeling - methods / Time Factors / Tissue Embedding - methods / Tissue Fixation - methods / Tissues