Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.
Bibliographical noteFunding Information:
This study was supported by the Brain Research Program through the National Research Foundation (NRF) funded by the Korean Ministry of Science, ICT, & Future Planning (NFR-2012M3A9C6049933 and NRF-2015M3C7A1028790). KBRI basic research program KBRI-2231-415. National Research Foundation of Korea (NRF-2015M3C7A1030964 and NRF-2015M3C7A1028396). We thank Dr. Hosung Jung for critically reading the manuscript. We also thank Carl Zeiss Inc. (Zena, Germany) for technical support utilizing the Z.1 light-sheet microscope.
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