Lee E , Choi J , Jo Y , Kim JY , Jang YJ , Lee HM , Kim SY , Lee HJ , Cho K , Jung N , Hur EM , Jeong SJ , Moon C , Choe Y , Rhyu IJ , Kim H , Sun W .

	Figure 1. Active Clarity Technique (ACT) is a rapid and efficient whole-brain clearing technique. (a) Workflow for clearing tissue with the ACT. The brain was isolated from a cardiac-perfused animal and post-fixed in 4% paraformaldehyde (PFA) overnight to induce DNA-protein crosslinking. The brain was immersed in a 4% acrylamide and thermal initiator-containing solution for 18–24 hours. The whole brain was incubated at 37 °C for 2 hours. After polymerization, lipid membranes were removed by ACT for 4–6 hours. (b) Diagram of the ACT-ECT system. (c) Brains before (left) and after (right) ACT processing; C57BL/6 mouse brains after polymerization, ACT, and refractive index matching. Scale bar, 1 cm. Injection of trypan blue into the ventricle of a cleared brain (bottom). Square unit; x: 5 mm, y: 5 mm.
	Active Clarity Technique (ACT) for three-dimensional imaging of protein and RNA distribution in adult brain samples. (a) Unsectioned mouse brain tissue image showing part of the midbrain stained with tyrosine-hydroxylase (20 × objective; stack size, 1,580 μm; step size, 2 μm). Scale bar, 100 μm. (b) Neurons labeled with the adeno-associated virus-green fluorescent protein (AAV-GFP) and immunolabeling of dendritic arbors with anti-GFP antibodies (10 × objective, 0.7 × confocal zoom; stack size, 1,180 μm; step size, 2 μm). Scale bar, 100 μm. (c) In situ hybridization of ACT-processed midbrain slice with 1.1 Kb DIG-labeled tyrosine hydroxylase (TH) probes. Scale bar, 100 μm. (d) Comparison of procedures and processing times between ACT and other clearing methods. (e) Images of brain blocks (1-mm thick) after processing. Dotted green lines indicate original sizes of blocks and red lines mark sizes after clearing. Square unit; x: 5 mm, y: 5 mm.
	Figure 3, Scalability of the Active Clarity Technique (ACT). (a) Rat brain hemisphere processed with 15 hours of ACT. Scale bar, 1 cm. (Right) Magnified fluorescent image of the boxed region in the middle (yellow). Nuclear-staining (SYTO16) of rat cerebellum (4 × 4 tile scan with the Fluar 5 × objective; stack size, 2,580 μm; stack step, 20 μm) acquired with the LSM 700 microscope. Scale bar, 1 mm. (b) Size comparison of an adult mouse brain and an adult rabbit brain (left). The rabbit brain was cleared after 36 hours of ACT (right). (c, d) Human spinal cord block cleared with ACT and immunolabeled. (c) Before and after ACT. Scale bar, 1 cm. Human spinal cord block (1.3–1.5 cm thick) was cleared (100 hours of ACT) and stained with SMI32 (red). SYTO16 (green) was used for nuclear staining (1.1 × confocal zoom; stack size, 1,020 μm; stack step, 1 μm). (d) Spinal cord block was immunostained for PGP 9.5 (cyan) and collagen type IV (red) (2 × confocal zoom; stack size, 420 μm; stack step, 5 μm). All three-dimensional reconstructed images were obtained with a Zeiss 780 (c) or 700 (d) confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens. Scale bar, 50 μm.
	Figure 4. Active Clarity Technique (ACT) for whole-organ clearing and immunolabeling. (a) Cleared organ samples (thymus, intestine, testis, lung, spleen, liver, and kidney) were treated with CUBIC-mount for 5 days to adjust the refractive indices of the tissue and reagent. Square units; x: 5 mm, y: 5 mm. (b) Transmission images of cleared whole organs (liver, lung, kidney, and spleen) by ACT. Images were taken after electrophoretic tissue clearing (ETC) in PBS. Details of the tissue architecture were visualized with a standard dissection microscope. Images were acquired on an Olympus BX53 digital microscope DP73 camera. Scale bar, 1 mm.
	ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) for rapid immunolabeling of dense tissues. (a) Comparison of diffusion rate using ACT-processed organs. (b) Schematic diagram for dense tissue immunohistochemistry. Tissues for centrifugal PRESTO (c-PRESTO) were centrifuged at 600 × g for 3 hours using a standard table-top centrifuge to expedite penetration of the primary and secondary antibodies. A syringe pump was used for the antibody reaction during syringe PRESTO (s-PRESTO). (c) Kidneys were labeled with collagen type IV using various protocols. Note that 3 hours of c- or s-PRESTO markedly enhanced the depth of specific labeling compared to that of the controls. Three-dimensional (3D) reconstructed images were obtained with a Zeiss 700 confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens, 2 × confocal zoom (stack size, 200 μm; stack step, 2 μm), and post-processed with Vaa3D software. Scale bar, 100 μm. Depth of fluorescence intensity was greater in PRESTO-treated tissue compared to that of free-diffusion labeled samples using ACT processed kidney tissue (mean ± standard deviation, n = 5). (d) Reconstituted 3D images of testis, lung, and liver. The organs were stained with acetylated tubulin (red in testis and lung) or laminin antibodies (red in liver). SYTO16 or DAPI were used for nuclear staining of the organs. Images were obtained with a Zeiss 700 confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens, 2 × confocal zoom (testis; stack size, 600 μm; stack step, 5 μm; liver; stack size, 226 μm; stack step, 2 μm; Scale bar, 100 μm), with a EC Plan-Neoflua 5 ×/0.16 M27 lens (lung; stack size, 1,265 μm; stack step, 5 μm; Scale bar, 500 μm).
	Supplementary Figure 6. Active Clarity Technique (ACT) is applicable to whole organisms (a)Whole organs were collected from 2 dayACT-processed whole mouse bodies. Scale bar, 1 cm. (b) Vertebrate and invertebrate model animals, such as chickens, Xenopus, and the small octopus were cleared by the ACT. Scale bar, 1 cm (see Supplementary Table 4 for ACT conditions and times).
	Figure 6. Whole body clearing of various organisms using the Active Clarity Technique (ACT).(a) Whole mouse body (3-weeks-old) was cleared in 24 hours using the ACT. After 3 days of incubation in CUBIC-mount solution, the ACT-processed mouse whole body became optically transparent and could be imaged without sectioning. Scale bar, 1 cm. (b) Image shows collagen type IV-labeled extracellular matrix (ECM) within the skull and underlying meninges. Images were obtained with a Zeiss 700 confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens, 2 × confocal zoom (stack size, 320 μm; stack step, 5 μm). Scale bar, 100 μm. (c) Comparison of optical transparency of whole zebrafish body before and after ACT clearing. Scale bar, 1 cm. (d) Whole rat embryo body (embryonic day 18) was cleared by the ACT. Scale bar, 1 cm. Embryo labeled with TrkA antibody shows the details of TrkA innervation in the foot. Imaged obtained with a Zeiss 700 confocal microscope with a Plan-Neofluar 5 ×/0.15 objective, 0.7 × confocal zoom (maximum projection; stack size, 1,250 μm; stack step, 15 μm). Scale bar, 500 μm.
	Figure 2. Active Clarity Technique (ACT) for three-dimensional imaging of protein and RNA distribution in adult brain samples.(a) Unsectioned mouse brain tissue image showing part of the midbrain stained with tyrosine-hydroxylase (20 × objective; stack size, 1,580 μm; step size, 2 μm). Scale bar, 100 μm. (b) Neurons labeled with the adeno-associated virus-green fluorescent protein (AAV-GFP) and immunolabeling of dendritic arbors with anti-GFP antibodies (10 × objective, 0.7 × confocal zoom; stack size, 1,180 μm; step size, 2 μm). Scale bar, 100 μm. (c) In situ hybridization of ACT-processed midbrain slice with 1.1 Kb DIG-labeled tyrosine hydroxylase (TH) probes. Scale bar, 100 μm. (d) Comparison of procedures and processing times between ACT and other clearing methods. (e) Images of brain blocks (1-mm thick) after processing. Dotted green lines indicate original sizes of blocks and red lines mark sizes after clearing. Square unit; x: 5 mm, y: 5 mm.
	Figure 3. Scalability of the Active Clarity Technique (ACT).(a) Rat brain hemisphere processed with 15 hours of ACT. Scale bar, 1 cm. (Right) Magnified fluorescent image of the boxed region in the middle (yellow). Nuclear-staining (SYTO16) of rat cerebellum (4 × 4 tile scan with the Fluar 5 × objective; stack size, 2,580 μm; stack step, 20 μm) acquired with the LSM 700 microscope. Scale bar, 1 mm. (b) Size comparison of an adult mouse brain and an adult rabbit brain (left). The rabbit brain was cleared after 36 hours of ACT (right). (c, d) Human spinal cord block cleared with ACT and immunolabeled. (c) Before and after ACT. Scale bar, 1 cm. Human spinal cord block (1.3–1.5 cm thick) was cleared (100 hours of ACT) and stained with SMI32 (red). SYTO16 (green) was used for nuclear staining (1.1 × confocal zoom; stack size, 1,020 μm; stack step, 1 μm). (d) Spinal cord block was immunostained for PGP 9.5 (cyan) and collagen type IV (red) (2 × confocal zoom; stack size, 420 μm; stack step, 5 μm). All three-dimensional reconstructed images were obtained with a Zeiss 780 (c) or 700 (d) confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens. Scale bar, 50 μm.
	Figure 5. ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) for rapid immunolabeling of dense tissues.(a) Comparison of diffusion rate using ACT-processed organs. (b) Schematic diagram for dense tissue immunohistochemistry. Tissues for centrifugal PRESTO (c-PRESTO) were centrifuged at 600 × g for 3 hours using a standard table-top centrifuge to expedite penetration of the primary and secondary antibodies. A syringe pump was used for the antibody reaction during syringe PRESTO (s-PRESTO). (c) Kidneys were labeled with collagen type IV using various protocols. Note that 3 hours of c- or s-PRESTO markedly enhanced the depth of specific labeling compared to that of the controls. Three-dimensional (3D) reconstructed images were obtained with a Zeiss 700 confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens, 2 × confocal zoom (stack size, 200 μm; stack step, 2 μm), and post-processed with Vaa3D software. Scale bar, 100 μm. Depth of fluorescence intensity was greater in PRESTO-treated tissue compared to that of free-diffusion labeled samples using ACT processed kidney tissue (mean ± standard deviation, n = 5). (d) Reconstituted 3D images of testis, lung, and liver. The organs were stained with acetylated tubulin (red in testis and lung) or laminin antibodies (red in liver). SYTO16 or DAPI were used for nuclear staining of the organs. Images were obtained with a Zeiss 700 confocal microscope with a Plan-apochromat 10 ×/0.45 M27 lens, 2 × confocal zoom (testis; stack size, 600 μm; stack step, 5 μm; liver; stack size, 226 μm; stack step, 2 μm; Scale bar, 100 μm), with a EC Plan-Neoflua 5 ×/0.16 M27 lens (lung; stack size, 1,265 μm; stack step, 5 μm; Scale bar, 500 μm).

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