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. 2022 Sep 12;13:5239. doi: 10.1038/s41467-022-32848-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Whole-body/organ tissue-clearing protocol with CUBIC reagents. Mice were sacrificed and perfused with 4% PFA. The fixed samples were subjected to CUBIC procedures. The basic protocol consists of fixation, delipidation with CUBIC-L, and RI adjustment with CUBIC-R (N). For 3D immunostaining, staining with washing is inserted before RI adjustment. Samples from the skin and peritoneum were embedded into 2% gelatin before RI adjustment. The images of the transparent samples are captured using light sheet fluorescent microscopy (LSFM). The images of the lung in each step are shown. b The 3D-reconstituted (3D) body image of a VE-cad-tdTomato mouse (female, 4 months). Before CUBIC procedures, tamoxifen (Tx) was injected (i.p.) for the induction of reporter. The enlarged images of the abdominal organs (liver, stomach, intestine and leg) are shown in the right panels. c The 3D images of the whole organs, including brain, lung, stomach, spleen, heart, intestine, and kidney in VE-cad-tdTomato mice (2–4 months) (Z; 10 μm step, digital zoom; brain, lung: 1.25, stomach, spleen, and kidney: 1.6, heart: 2.5, intestine: 2.0). d The enlarged 3D images (white insets) (kidney and stomach) of (c). e The 2D (XY) images of the spleen, kidney, and intestine. White insets are magnified next to each image. f The 3D image of the kidney and 2D image of the intestine in tomato-lectin-injected mice (C57BL/6J, 5w). The mouse was sacrificed 5 min after inoculation of Texas-Red conjugated with tomato-lectin (Z; 10 μm step, digital zoom; kidney: 2.0, intestine: 1.6). g The 3D images of the blood vessels in the skin and peritoneum embedded in gelatin. The skin and peritoneum samples from a VE-cad-tdTomato mouse (3 months) were embedded in 2% gel before RI adjustment. White insets are magnified next to each image (Z; 10 μm step, digital zoom; 2.0).