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Insights tissue clearing

Published on Imaging & Microscopy (
Mar. 16, 2017

Today, imaging entire cleared samples is a vital necessity to understand biological systems and processes. Several protocols have been published describing different clearing protocols. The article will address some of the frequently used techniques.

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Frequently asked questions

Can I use DAPI? - DAPI is excited with 405 nm. This is a pretty short wavelength which is scattered and absorbed by the tissue. It is absolutely not suitable for deep imaging. One should try DRAQ5™ or TO-PRO®.

My sample contains GFP, which clearing should I try? - You may try uDISCO, ethyl cinnamate, CUBIC, CLARITY, FluoClearBABB or iDISCO. iDISCO maybe of advantage due to the fact that you can label it with a far red dye for a better penetration of the excitation wavelength.

The tissue I am working with is very dense. What clearing is capable to get it transparent? - Users working with dense samples often use BABB, 3DISCO or iDISCO+.

The sample I would like to image is colorized (e.g.: liver). How can I cope with colorization? - You may try iDISCO+ or CUBIC-perfusion protocol. These protocols were designed to decolorize sample tissue.

The sample I would like to image is very small. How can I mount it? - If you work with organic solvents, you may prepare an empty but cleared cube with 1% low melting agarose. After clearing the cube you make a small cut on top of it where you insert your cleared and stained sample. One can also directly embed sample into agarose and do clearing of the sample within the agarose. In that case extend dehydration (100% over night) so that the agarose is completely dry. If there is only a small amount of water remaining in the cube it will start shrinking and it will get milky as soon as it is transferred to the clearing solution. If you work with aqueous buffers you may insert sample into a FEP tube or a cube of Phytagel™ (Sigma-Aldrich Co. LLC.).

I cleared my sample but it is still not transparent and shows a strong background. -If you have applied an aqueous buffer based protocol you may want to try an organic solvent protocol. Methanol treatment might reduce the background. Please consider to dehydrate the sample completely. If used dehydration solution (tetrahydrofuran, ethanol or methanol) is stored in a bottle which is opened frequently, there already will be a remarkable amount of water in that solution due to hygroscopic characteristic of the solvent.

I cleared my sample using the CLARITY protocol. What kind of solution should I use for imaging? - Some CLARITY imaging solutions are very expensive. To reduce costs you may try sRIMS, 63% thiodieethanol or 80% glycerol. The iDISCO protocol resulted in a sample with a strong labelling only on the surface. What should I do? - Increase the dilution of the antibody and find further information at:

My sample is very soft. How can I mount it for the image acquisition? - You may try some glue. Loctite Professional®, krazy glue® or picodent® are even stable in organic solvents.

I would like to label my sample with fluorophores, which are suitable? - Every fluorophore with an excitation maximum between 500 nm and 785 nm is suitable. The extended wavelength can penetrate tissue better. DiI or DiAsp are not compatible with most clearing protocols. Membranes as binding site for these dyes are often altered or affected by the clearing. For example users have good results with using Alexa Fluor® 647, Alexa Fluor® 750 (Thermo Fisher Scientific Inc.), ATTO 647 (ATTO-TEC GmbH), VivoTag® 680, VivoTag® 750 (PerkinElmer Inc.), Cy7, or IRDye ®(LI-COR, Inc.).



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