Background

Optimization of tissue imaging techniques had to overcome several inherent problems, including lack of optical tissue transparency and spatial limits on antibody penetration. The solutions to these problems have evolved over time. Major advances in microscopy provided for superior 2D and 3D image resolution (Richardson and Lichtman, 2015; Whitehead et al., 2017). Thin sections, less than 40 microns in thickness, allowed most of the antibodies to reach their target proteins. Combined with diverse fluorescent tags, these antibodies revealed tissue complexities beyond those known before. However, evaluation of a tissue block or whole organ divided into many thin sections remained far from trivial. Following the time-consuming process of cutting and staining individual sections, an inherent discontinuity of specimens required the development of sophisticated imaging reconstruction techniques for accurate quantification of cells and ability to follow their individual projections, e.g., micro-optical sectioning tomography (MOST) (Li et al., 2010). Finally, to increase tissue transparency, a number of strategies to “clear” the tissue has been proposed, first by Werner Spalteholz as early as 1914 (Spalteholz, 1914) and by many other researchers thereafter (Efimova and Anokhin, 2009; Hama et al., 2011; Ertürk et al., 2012; Ke et al., 2013; Susaki et al., 2014; Fumoto et al., 2016). Despite the effectiveness of these strategies, there were limitations in tissue types and species in which they performed best, with some causing tissue shrinking (for review, Mano et al., 2018). Other technical difficulties include the rate and volume at which antibodies penetrated into a thick cleared tissue block or section, or whole organ specimen for labeling of proteins, and adverse effects of time and/or exposure to light on fluorescence emission.

In 2013, Stanford researchers Kwanghun Chung and Karl Deisseroth developed a novel approach called CLARITY (Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tis-sue-hYdrogel) (Chung et al., 2013; Chung and Deisseroth, 2013). By simultaneously removing lipids and infusing the entire protein structure with a hydrogel, CLARITY preserved the tissue architecture, proteins and nucleic acid molecules, while making a large tissue block or an entire organ optically transparent. Importantly, the removal of lipids using this method enhanced antibody penetration into the preserved tissue, facilitating immunohistochemical staining, allowing for more efficient and accurate quantitative analysis. The success of CLARITY is highlighted by its increasing popularity among neuroscientists and biologists studying diverse tissues and organs (Azaripour et al., 2016; Mortazavi et al., 2016; Jensen and Berg, 2017; Vigouroux et al., 2017; Du et al., 2018; Yu et al., 2018).

Active use of CLARITY technique resulted its further optimization, including PACT (passive CLARITY technique) and PARS (perfusion assisted agent released in situ), or ACT-PRESTO (active cleaning technique pressure related efficient and stable transfer of macromolecules into organs) (Yang et al., 2014; Tomer et al., 2014; Lee et al., 2016). These methods proved to be applicable to a diverse array of tissues, including the peripheral organs such as the liver, kidney, intestine and lung (Lee et al., 2014; Font-Burgada et al., 2015; Neckel et al., 2016; Saboor et al., 2016). While there are common features in CLARITY methodology, the processing and imaging of diverse tissues, organs or whole animals may differ between model organisms. Human tissues also require special considerations due to the high lipid content of human brain tissue, and often the prolonged post-mortem interval (PMI) that can affect the quality of tissue, and its fixation.

Here we share our protocols for using CLARITY to visualize a number of proteins of interest in brain tissue of several species, including zebrafish, rat, mouse, rhesus monkey, and human. We find the technique to be relatively simple to execute, highly efficient in clarifying whole zebrafish, individual brains, large brain tissue blocks or thick sections. We also find that our CLARITY protocol allows for using lower than earlier reported antibody concentrations to effectively reveal target proteins, enabling high-quality 3D visualization. In addition to earlier proposed semi-quantitative analysis of CLARITY-processed whole-brain zebrafish samples, based on fluorescence Intensity (Lindsey and Kaslin, 2017), we show that 3D analytical tools (e.g., Fiji or Imaris) can provide accurate counts and morphological parameters of labeled cells, axons, dendrites, or any other quantitative immunohistochemical labeling. Together, we find CLARITY to be an exceptional tool for 3D visualization and quantification of brain tissue constituents, which can further be used in studies of neurogenesis, connectivity, and pathological brain conditions.