Tissue clearing as the name would suggest, refers to rendering tissues transparent with the use of various chemicals and techniques. A major advantage of tissue clearing is the ability for researchers to preserve spatial resolution. Whereby traditional histology methods involve cutting sample into thin slices and possibly damaging the morphology. There are various methods and techniques, and this article will discuss the difference between the two. At the microscopic level, tissues are composed of lipid, protein, carbohydrate, minerals and more, each with varying refractive indexes. A refractive index relates to how light is bent going through a medium. In context, the cell’s refractive index is related to its mass distribution and provides insight for diverse biological models. Tissue clearing is thought to work by matching the refractive index of each the cellular components to that of its environment or solvent. On the premise that objects with similar refractive indices will become transparent because light is being bend at similar angles.

Two different approaches to discuss are protein hyper-hydration and solvent high refractive index matching. In both cases, while the exact details of how these clearing reagents work are not fully know, scientists believe that it revolves around protein and solvent interaction. Protein hyper-hydration, or aqueous clearing, works by lowering proteins’ refractive index to that of water. The clearing reagent enters the cell, encapsulates the proteins and by doing so lowers the RI. A key advantage of this method is its compatibility with fluorescent protein. Solvent high refractive index matching, or solvent clearing, works by raising the refractive index by removing the lipids and then matching the RI of the proteins left behind. The sample is first dehydrated in an alcohol to remove the water, then the clearing reagent is introduced. It is thought that the clearing reagent removes the lipids. Replacing the lipids in the cells with a solvent that matches the refractive index of the proteins is how the clearing phenomena is seen. A key advantage to this is the immunolabeling and IHC compatibility. However, a large concern for this technique is the morphological altercations and the inability of lipid preservation.

With the exception being Visikol Histo. With the defining characteristic being its ability to preserve the histological integrity of cleared specimens so that clearing can be reversed. The reversibility of Visikol HISTO means you can validate 3D data against slide-based histopathology. Visikol offers a suite of clearing reagents – Histo I, II, M. All of which are compatibility with immunostaining, fluorescence proteins and non-toxic. After clearing a tissue with Visikol HISTO, one can wash the solution back out of the tissue, and rehydrate it, restoring the tissue to its original state. The Visikol HISTO approach has been designed to pair with immunolabeling and is reversible so that 3D histology can be followed up with traditional 2D histology.

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