T-CLEARE: A Pilot Community-Driven Tissue-Clearing Protocol Repository

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 many articles do not provide sufficient detail to replicate or reproduce experimental results. 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 provide a forum for the community to share evaluations and modifications of tissue-clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.


INTRODUCTION
Tissue clearing refers to methods that increase the physical transparency of biological 31 samples. Through a series of chemical or physical steps, tissue-clearing methods homogenize the 32 refractive index of a sample by removing, replacing, or modifying light-scattering components . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 12, 2023. ; https://doi.org/10.1101/2023.03.09.531970 doi: bioRxiv preprint 49 parameters and research goals (34). Published articles and protocol repositories, such as 50 protocols.io, usually report protocols after they have been optimized and do not provide lessons 51 learned and modifications that were required to generate a successful result.

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This article describes the development and implementation of T-CLEARE (Tissue 53 CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE is intended to 54 complement publications and protocol repositories and help the community compare and 55 evaluate protocols when designing a tissue-clearing strategy. T-CLEARE is based on the results . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 12, 2023. ; https://doi.org/10.1101/2023.03.09.531970 doi: bioRxiv preprint 4 56 of a survey that asked researchers to identify tissue-clearing protocols they have used.
57 Respondents were also asked to provide details not usually found in publications or protocol 58 repositories, including protocol effectiveness and ease of use, modifications needed to optimize a 59 protocol for specific circumstances, pre-and postprocessing steps not specific to published 60 protocols, and negative results (i.e., instances when a tissue-clearing protocol did not perform as 61 expected or was not appropriate for the application). T-CLEARE represents each tissue-clearing 62 protocol reported by the community as a flow chart of five steps: fixation, 63 pretreatment/decolorization, labeling, delipidation, and refractive index matching. Each flow 64 chart is included in a tree diagram and organized according to whether it was very successful, 65 successful, or not successful. Users can filter protocols based on whether it was successful, the 66 tissue and organism studied, and the details of five different tissue-clearing steps.

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Tissue-Clearing Survey.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 12, 2023. ; https://doi.org/10.1101/2023.03.09.531970 doi: bioRxiv preprint 6 102 from a published protocol or specifying modifications using the "other" boxes ( Figure 1). 103 Respondents were asked to provide a citation (e.g., publication reference, DOI) for any details in 104 the "other" boxes because these represented modifications to one of the protocols listed or use of 105 a protocol not included in the table. In addition, respondents were asked a range of question to 106 capture additional details about their experience ("Was the protocol successful?", "Why was the 107 protocol chosen?", "Are you still looking for better tissue-clearing protocols?") and their 108 experiment (e.g., specimen type, tissue type, feature of interest, label type, type of analysis, level 109 of analysis). A total of 29 respondents provided feedback.

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Each unique tissue-clearing protocol was diagramed as a flow chart using Lucidchart 121 software (Figure 2). Each flow chart included reported details for five major tissue-clearing 122 steps) and the organism and tissue being cleared. Each protocol flow chart was organized 123 according to whether the respondent indicated the protocol was very successful, successful, or 124 not successful. 167 and organism and tissue type (e.g., mouse-whole brain; mouse-cochlea). These filters can help . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 12, 2023. ; https://doi.org/10.1101/2023.03.09.531970 doi: bioRxiv preprint 9 168 users tease how successful a method or group of methods, is for a given tissue. For example, 169 users can select "successful" protocols that follow hyperhydrating principles for fixation, 170 pretreatment, labeling, and refractive index matching in "mouse brain."

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In summary, T-CLEARE is a novel, web-based repository of tissue-clearing protocols to 221 help the community share, find, and evaluate available tissue-clearing protocols in real time. As 222 more information is added, we hope that T-CLEARE can help the community coalesce around 223 reporting standards and standard tissue-clearing protocols. These community standards will help 224 ensure consistent data collection and reporting, improve data interpretation, and facilitate data 225 sharing among the scientific community. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 12, 2023. ; https://doi.org/10.1101/2023.03.09.531970 doi: bioRxiv preprint