Abstract
Histological analysis has been largely confined to two-dimensional analysis of thin tissue sections, hampering detailed understanding of three-dimensional cellular distribution in biological tissues. Tissue optical clearing methods enable three-dimensional histological analysis by rendering tissues transparent and suitable for microscopic detection of the fluorescent signals inside. Despite their great potential in histological research, the tissue clearing methods are not readily accessible to many researchers because of hazardous chemicals, complicated protocols and advanced microscopy. Furthermore, poor antibody penetration represents an additional major obstacle when performing three-dimensional immunohistochemical studies. Here, we have examined tissue optical clearing of a mouse brain slice by a non-hazardous aqueous solution, ScaleA2. We modified the ScaleA2 solution by increasing the concentration of detergent. A simple immersion in the modified ScaleA2 solution alone enabled highly intense and uniform immunolabeling into deep tissues in three-dimensional immunostaining. Conventional confocal microscopy could image three-dimensional immunostaining of vasculature and astrocytes with fine processes to 1 mm imaging depth. Collectively, our technically straightforward clearing method will facilitate the common application of three-dimensional immunohistochemical analysis in many research fields including neuroscience, expanding our understanding of the detailed spatial cellular distribution underlying the physiology and pathology.
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All applicable institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution.
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418_2017_1614_MOESM1_ESM.tif
Online Resource 1 The cross-sectional images of a mouse brain slice without tissue clearing, followed by immunostaining with anti-GFAP antibody (cyan) and nuclear staining dye SYTO16 (green). Two-mm-thick mouse brain coronal slice was immunostained and excised at 2 mm inside from the surface to study antibody penetration at the cross-section by confocal laser scanning microscopy with a 10x objective lens. Less immunolabeling of GFAP was detected especially in corpus callosum. The x-axis represents the tissue depth. CC: corpus callosum. Scale bar: 200 μm. Supplementary material 1 (TIFF 3211 kb)
418_2017_1614_MOESM2_ESM.mp4
Online Resource 2 Animation (rotation and zooming in) of 3D image in mouse hippocampus. A mouse brain slice cleared with ScaleA2-T followed by immunostaining with anti-GFAP antibody (cyan, astrocytes), tomato lectin (red, blood vessels) and SYTO16 (green, nuclei) was imaged three-dimensionally by confocal microscopy with a 10x objective lens to 1 mm imaging depth (Fig. 3). Scale bar: 200 μm. Supplementary material 2 (MP4 7820 kb)
418_2017_1614_MOESM3_ESM.mp4
Online Resource 3 Animation (rotation and zooming in) of higher magnified 3D image in mouse hippocampus. A mouse brain slice cleared with ScaleA2-T followed by immunostaining with anti-GFAP antibody (cyan, astrocytes), tomato lectin (red, blood vessels) and SYTO16 (green, nuclei) was imaged three-dimensionally by confocal microscopy with a 20x objective lens to 400 μm imaging depth (Fig. 4). Scale bar: 50 μm. Supplementary material 3 (MP4 7668 kb)
418_2017_1614_MOESM4_ESM.mp4
Online Resource 4 Animation (rotation) of 3D image in mouse substantia nigra. A mouse brain slice cleared with ScaleA2-T followed by immunostaining with anti-TH antibody (red, dopaminergic neurons) and SYTO16 (green, nuclei) was imaged three-dimensionally by confocal microscopy with a 10x objective lens to 550 μm imaging depth (Fig. 5). Scale bar: 200 μm. Supplementary material 4 (MP4 1473 kb)
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Kakimoto, T. Validation of an easily applicable three-dimensional immunohistochemical imaging method for a mouse brain using conventional confocal microscopy. Histochem Cell Biol 149, 97–103 (2018). https://doi.org/10.1007/s00418-017-1614-0
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DOI: https://doi.org/10.1007/s00418-017-1614-0