T-CLEARE: a pilot community-driven tissue clearing protocol repository

Abstract

Selecting and implementing a tissue clearing protocol is challenging. Established more than 100 years ago, tissue clearing is still a rapidly evolving field of research. There are currently many published protocols to choose from, and each performs better or worse across a range of key evaluation factors (e.g., speed, cost, tissue stability, fluorescence quenching). Additionally, tissue clearing protocols are often optimized for specific experimental contexts, and applying an existing protocol to a new problem can require a lengthy period of adaptation by trial and error. Although the primary literature and review articles provide a useful starting point for optimization, there is growing recognition that results can vary dramatically with changes to tissue type or antibody used. To help address this issue, we have developed a novel, freely available repository of tissue clearing protocols named T-CLEARE (Tissue CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE incorporates community responses to an open survey designed to capture details not commonly found in the scientific literature, including modifications to published protocols required for specific use cases and instances when tissue clearing protocols did not perform well (negative results). The goal of T-CLEARE is to help the community share evaluations and modifications of tissue clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.

Keywords: database; microscopy; neuroscience; tissue clearing; tissue clearing methods.

FIGURE 1

Table of commonly used tissue clearing protocols included in the Tissue Clearing Survey (https://doryworkspace.org/tissue_clearing_feedback). Respondents identified the tissue clearing protocols used by checking boxes in the table or by entering details in the “Other, specify” column. The table demonstrates an example tissue clearing protocol response based on iDISCO with a modification to the labeling step indicated in the “Other, Specify” column.

FIGURE 2

Example flow charts created from reported tissue clearing protocols. (A) Example flow charts of successful tissue-clearing protocols. The tissue cleared is indicated in the right-most box. (B) Example flow charts of not successful tissue-clearing protocols. The tissue cleared is indicated in the right-most box.

FIGURE 3

Screenshot of T-CLEARE user interface. Boxes in light blue, aqua, and light red indicate whether the mode of action for that step is based on hyperhydrating solutions/hydrophilic, tissue transformation/hydrogel, and organic solvents/hydrophobic, respectively. Yellow boxes indicate the fixation step that can be classifed according to more than one mode of action. Gray boxes indicate the respondent entered text in the “Other, specify” in the survey table.

FIGURE 4

Organisms and tissues in reported tissue clearing protocols.

FIGURE 5

Reported tissue clearing protocols used without modification. iDISCO, immunolabeling-enabled three-Dimensional Imaging of Solvent-Cleared Organs (Renier et al., 2014), PACT, PAssive Clarity Technique (Yang et al., 2014), CUBIC, Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (Tainaka et al., 2014), BABB, Benzyl Alcohol/Benzyl Benzoate (Dodt et al., 2007), PEGASOS, polyethylene glycol (PEG)-associated solvent system (Jing et al., 2018), SHORT, SWITCH - H2O2 - antigen Retrieval (Pesce et al., 2022).

FIGURE 6

Overall success in reported tissue clearing protocols. Individual protocols in responses have been grouped according to mode of action (Organic Solvents/Hydrophobic, Hyperhydrating Solutions/Hydrophilic, Tissue Transformation/Hydrogel, Other).

FIGURE 7

Reported tissue clearing success by respondent job title.