[RETRACTED] Whole-Body Expansion Microscopy with Immunofluorescence and Histological Stains (WB-ExM-IF & WB-ExM-Histo) v1

[RETRACTED] The interpretation of Whole Body Imaging (WBI) data necessitates comprehensive anatomical knowledge to accurately determine cell-type identity; however, resources pertaining to the internal anatomy of larval zebrafish are limited. To mitigate this gap, we established an advanced Whole Body Expansion-Microscopy (WB-ExM) protocol, facilitating the acquisition of molecular and cell-type information at subcellular resolution throughout the entire organism. While high-quality resources are available for the larval zebrafish brain1,2,3 there remains a deficiency in materials concerning its visceral anatomy. In order to supplement existing histological, X-ray, and Expansion Microscopy methods aimed at mapping body-wide anatomy4,5,6,7 and to develop a technique that yields high-fidelity molecular and anatomical data with enhanced processing speeds and compatibility with older samples, we devised an enzyme-free, rapid, and robust whole-body expansion microscopy method8,9, effective on larvae up to at least 14 days post-fertilization. This method involves the use of high-temperature (100°C) chemical hydrolysis to uniformly soften tissues, embedding within a medium-density gel with reduced protein-gel anchoring, and repeated embedding post-digestion with moderate ~1.5x expansion factors in each cycle, culminating in a robust and uniform expansion even of challenging structures such as cartilage embedded in soft tissue. This protocol results in excellent optical clearing, retains high levels of antibody signals. A demo dataset can be viewed here. Dorsal view of a double transgenic zebrafish animal labeling the ventricular and vascular systems (Tg(foxj1a:eGFP) x Tg(flk1:dsRed-CAAX)), stained against eGFP (magenta) and dsRed (green) (10 days post-fertilization, expanded ~2×). 1. Kunst, M. et al. A Cellular-Resolution Atlas of the Larval Zebrafish Brain. Neuron 103, 21-38.e5 (2019). 2. Randlett, O. et al. Whole-brain activity mapping onto a zebrafish brain atlas. Nat Methods 12, 1039–1046 (2015). 3. Tabor, K. M. et al. Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping. 4. Copper, J. E. et al. Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 208, 38–46 (2018).5. Ding, Y. et al. Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography. eLife 8, e44898 (2019). 6. Steib, E. et al. TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy. Cell Reports Methods 2, 100311 (2022). 7. Sim, J. et al. Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy. Preprint at https://doi.org/10.1101/2021.05.18.443629 (2021). 8. Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy.9. Wang, Y. et al. EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell 184, 6361-6377.e24 (2021).