Tissue clearing guide
Learn everything you need to know about the tissue clearing technique, including hydrophobic, hydrophilic, and hydrogel-based approaches.
Download our tissue clearing methods poster
What is tissue clearing?
Tissue clearing is a process that makes tissue samples transparent by removing opaque molecules. This technique minimizes light scattering and light absorption to enable better light penetration during microscopy1. The resulting minimization of light absorption in the sample allows the generation of 3D microscopy images that depict biological structures more accurately compared to conventional 2D methods. Advanced 3D microscopy, in combination with rapidly advancing tissue-clearing techniques, facilitates high-resolution optical exploration of intact tissues, organs, and even whole organisms, illuminating subcellular details in whole organs and even small mammals.1
NeuN antibody (ab177487) (green) and DAPI (blue) used with our Tissue Clearing Kit (ab243298) to clear and stain a 1 mm thick coronal section of mouse brain.
Further insights can be gained by immunolabeling various targets in cleared tissue samples. This enables visualization of sub-cellular structures and proteins while preserving their spatial distribution within the specimen. This provides an improved representation of the in vivo microenvironment of the specimen, allowing for better prediction and research of targets of interest2,3,4,5.
Why clear 3D cell cultures
3D cell culture models (eg organoids, microtissues, spheroids) give a much better representation of the in vivo microenvironment and thus offer improved predictive capability compared to traditional 2D cell culture models. However, one of the problems with 3D cell culture models has been their characterization. Assays that rely on the dissolution of the cells in a 3D structure lose the spatial features that make these models so valuable.
Despite 3D cultures being relatively thin (compared to a whole organ), light still attenuates after 1–3 cell layers and thus most imaging techniques (widefield, confocal) characterize only the cells on the periphery of these models. Since peripheral cells receive the highest levels of compounds and nutrients, they do not represent the whole model. Peripheral cells often exhibit atypical responses to compounds than the average cell within the model. While optical sectioning solves the issue of light attenuation and scattering, this only provides information about single planes of a 3D model.
With tissue clearing methods, it's possible to imagine the entire 3D cell culture and simultaneously preserve structural integrity.
Albumin antibody (ab207327) (yellow), CD68 antibody (green), Vimentin antibody (red) and DAPI (blue) used with 3D Cell Culture Clearing Kit (ab243299)to clear and stain a 3D hepatocyte cell culture spheroid.
Tissue clearing methods and tips
The clearing of tissues and even whole organs is achieved through several chemical and (in some cases) electrophoretic steps. Tissue clearing is conducted by incubating the specimen in a series of specialized solutions to eliminate interfering molecules such as lipids, pigments, and calcium phosphate. There are three prevalent approaches to tissue clearing: hydrophobic techniques employing organic solvents, hydrophilic techniques using aqueous solutions, and hydrogel-based methods. Each approach has its own advantages and limitations. For an in-depth look at these, you may find it useful to browse the protocols and FAQs of the main types (Table 1).
Table 1. Some of the main tissue clearing methods and their associated online resources.
Hydrophobic tissue clearing
Hydrophobic tissue clearing processes use dehydration steps to eliminate water, followed by organic solvents to remove most lipids. This minimizes light scattering by matching the refractive index of the tissue1. Hydrophobic tissue clearing offers a straightforward approach, requiring only the sequential immersion of specimens in various solutions.
A popular approach to hydrophobic tissue clearing is the DISCO framework (3D imaging of solvent-cleared organs), a versatile technique which can easily be tailored to diverse experimental requirements2-4 . The DISCO framework seamlessly integrates with immunolabeling methodologies across various tissue types and species, making it an ideal method to study intricate biological structures.
The initial dehydration step can cause shrinkage of the tissue, while the organic solvents permanently preserve the tissue. This facilitates multiple imaging sessions and extended sample reanalysis, particularly through immunolabeling techniques that can permanently stabilize endogenous fluorescent signals.
Related products
Our portfolio includes tissue clearing reagents developed with Visikol Inc, who are experts in digital pathology, 3D cell culture assays and 3D tissue imaging, and developed the Visikol® HISTO™ tissue clearing technology which is the basis of our reagents.
- 3D Cell Culture Clearing Kit
Hydrophilic tissue clearing
Hydrophilic tissue clearing approaches use water-soluble reagents to maintain the tissue's native hydrophilic properties and prevent shrinkage. These hydrophilic reagents form hydrogen bonds with molecules like proteins; this bond formation helps preserve the 3D structure of tissue components, ensuring the integrity of fluorescent labeling. Additionally, these reagents can serve as refractive index-matched media1.
Ueda and colleagues identified a range of amino alcohols with delipidation and decolorization capabilities, leading to the development of the CUBIC method (brain or body imaging cocktails and computational analysis). The removal of lipids enables improved penetration of molecules like antibodies into the tissue allowing for improved immunolabeling in large samples1, making the CUBIC method a powerful tool for imaging cleared tissue2-5 .
Continued refinement of the CUBIC approach has led to enhanced performance tailored to specific experimental requirements. For example, the CUBIC-X protocol uses imidazole and antipyrine as hyperhydrating reagents, causing sample expansion. This approach has been used successfully in expansion microscopy, enabling single-cell resolution in adult mouse brain imaging6.
Related products
We offer a range of products specifically designed for tissue clearing using CUBIC technology.
- 3D Tissue Staining Kit – CUBIC
- 3D Tissue Staining Pots (x10)
Hydrogel-based tissue clearing
Hydrophilic tissue clearing approaches use water-soluble reagents to maintain the tissue's native hydrophilic properties and prevent shrinkage. These hydrophilic reagents form hydrogen bonds with molecules like proteins; this bond formation helps preserve the 3D structure of tissue components, ensuring the integrity of fluorescent labeling. Additionally, these reagents can serve as refractive index-matched media1.
Ueda and colleagues identified a range of amino alcohols with delipidation and decolorization capabilities, leading to the development of the CUBIC method (brain or body imaging cocktails and computational analysis). The removal of lipids enables improved penetration of molecules like antibodies into the tissue allowing for improved immunolabeling in large samples1, making the CUBIC method a powerful tool for imaging cleared tissue2-5 .
Continued refinement of the CUBIC approach has led to enhanced performance tailored to specific experimental requirements. For example, the CUBIC-X protocol uses imidazole and antipyrine as hyperhydrating reagents, causing sample expansion. This approach has been used successfully in expansion microscopy, enabling single-cell resolution in adult mouse brain imaging6.
Related products
We offer a range of products specifically designed for tissue clearing using CUBIC technology.
References
- Ueda, H. R.,, Ertürk, A.,, Chung, K.,, et al. Tissue clearing and its applications in neuroscience Nature Reviews Neuroscience 21 ,61-79 (2020)
- Susaki, E. A.,, Tainaka, K.,, Perrin, D.,, et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157 ,726-739 (2014)
- Cai, R.,, Pan, C.,, Ghasemigharagoz, A.,, et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nature Neuroscience 22 ,317-327 (2019)
- Tainaka, K.,, Kubota, S. I.,, Suvama, T. Q.,, et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159 (4),911-924 (2014)
- Belle, M.,, Godefroy, D.,, Couly, G.,, et al. Tridimensional visualization and analysis of early human development. Cell 169 (1),161-173 (2017)
- Murakami, T. C.,, Mano, T.,, Saikawa, S.,, et al. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nature Neuroscience 21 ,625-637 (2018)