Fig. 2: Development of a versatile protocol for whole-organ/body dye staining.

a Schematic of the surrogate assay. A cylinder-shaped, fixed gelatin gel containing DNA or rabbit immunoglobulin was used to experimentally evaluate the 3D staining patterns in the interior of the gel (rimmed or gradual patterns). b Representative results of DNA-containing gelatin gel staining with propidium iodide (PI), an ionized, lipophobic, and cell-impermeable nuclear stain. A high concentration of salt modulated the gel–stain interaction, leading to the improvement of the interior staining patterns. Scale: 1 mm. c Representative results of DNA-containing gelatin gel staining with SYTO 16, a lipophilic and cell-permeable nuclear stain. Additional use of ScaleCUBIC-1A (SC-1A) chemicals in high-concentration salt conditions improved the penetration patterns with decreased signal intensities (indicated as “4× enhanced”). Scale: 1 mm. The profiles in b and c show the mean intensity ± SD of the six diameter regions as in a. d, e The results in b and c were replicated by using the fixed and delipidated tissues (cerebellar hemispheres). After 3D staining, the samples were sectioned to evaluate the infiltration of the stain inside the tissue. The sections were also restained with DAPI to check the detectability of the nuclei (post 2D). Scale: 1 mm. f–h The interaction-modulated 3D staining was scaled up to a whole infant marmoset body. f The cleared and PI-stained infant marmoset body. PI staining was performed under a high ion strength condition (see “Methods”). Scale: 1 cm. g The reconstituted whole-body 3D image acquired with a custom-built LSFM (voxel size: 10.3 × 10.3 × 100 μm³) after staining and clearing. White boxes indicate the positions of the reconstituted x–z images in the panels on the right. Scale: 1 cm. h The reconstituted x–z images at the indicated positions in g. L left, R right. Scale: 2 mm.