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Set up rapid tissue clearing for 3D tissue imaging using simple protocols and standard laboratory equipment.
Clearing allows you to image thicker tissue sections, or even whole organs, by increasing tissue transparency, which ultimately yields more data from each experiment. Here we go through some tips, techniques, and reagents for tissue clearing.
Tissue clearing techniques all aim to make tissues or cell cultures more transparent to overcome their opacity, which prevents them from being penetrated by visible wavelengths of light under the microscope. Rather than going through the laborious process of making very thin sections of a tissue, which can disrupt morphology, it can be preferable to clear tissue through several chemical steps. Clearing also opens up the option to 3D microscopy to generate images more reflective of biological structures than those seen with conventional 2D methods.
Clearing methods typically rely on immersing the tissue in different solutions (like an organic solvent or an aqueous solution with a high refractive index) or possibly embedding the tissue in a hydrogel before extracting tissue lipids with detergents. Different methods have an array of benefits and limitations, depending on the type of tissue you use.
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
There are multiple clearing methods, each with their own benefits 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.
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To learn more about what tissue clearing is, the various types of clearing methods available, how best to make use of it, watch this webinar split into four short sections (click three-line button on the player to access different parts):
- Introduction to methods for tissue clearing
- Optimizing tissue thickness and antibody concentration
- Considerations for direct and indirect labeling, auto-fluorescence, and multi-color labeling
- Imaging cleared tissues
Tissue clearing reagents and kits
Recently developed tissue clearing methods, such as CLARITY, CUBIC, i/3/u/vDISCO, etc, have been used to produce beautiful images that push the boundaries in fields such as neuroscience and developmental biology. To streamline this process, we have developed tissue clearing kits and reagents so that you can quickly clear tissues or 3D cell cultures. The protocols are simple, use standard laboratory equipment, and are compatible with immunostaining, fluorescent proteins, and chemical dyes. Clearing is reversible so you can section your tissue for conventional H&E or IHC staining after 3D imaging.
The 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.
You can get tissue clearing kits validated with antibodies for essential markers, such as NeuN, GFAP, and Iba1. We test these antibodies on 1 mm mouse brain sections, neuronal spheroids, and hepatic spheroids (depending on the marker).
- Tissue Clearing Kit (ab243298)
- 3D Cell Culture Clearing Kit (ab243299)
You can also access a complete Visikol® HISTO™ protocol for tissue clearing and labeling, here.