Sample Preparation


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Clearing tissue samples using CLARITY begins with introducing hydrogel monomers into the tissue. The monomers are introduced via transcardial perfusion and/or sample incubation with a hydrogel monomer solution.


CLARITY-applicable Tissue Samples

CLARITY can be performed on both freshly-extracted tissue samples as well as fixed tissue samples. The main difference in processing these samples is the incubation time in the hydrogel monomer solution.

While CLARITY was developed for brain tissue, it is applicable to a variety of other tissues as well. Challenges may arise for tissues that are particularly fibrous as they may undergo increased crosslinking and therefore limit the hydrogel porosity during clearing. CLARITY uses fat-collecting micelles to remove light-scattering lipids from the tissue to achieve transparency. If there are other non-fatty light-scattering elements in the tissue, additional clearing using a separate solution to remove those elements may be needed.

Steps for Sample Preparation

Make hydrogel solution

Begin by preparing the hydrogel solution. This solution can be made in batches and stored in the freezer until needed.

Complete perfusion

Removal of blood cells in the tissue before hydrogel embedding is important. Otherwise, they will be incorporated into the hydrogel matrix and could act as a source of autofluorescence during imaging. Transcardial perfusion using PBS and the hydrogel solution (in place of plain fixative solution) is therefore highly recommended for animal tissue samples if possible. In addition to removing blood cells, perfusion with the hydrogel solution introduces hydrogel monomers inside the tissue. This aids in reducing the sample incubation time during which monomers in the solution outside the tissue passively diffuse in.

If perfusion is not feasible or the tissue is already fixed, the sample should be placed directly into the hydrogel solution for incubation. If autofluorescence is a problem, these samples may require several washes before sample incubation to first rinse out the blood cells.

Incubate sample in hydrogel solution

Following perfusion, place the sample in hydrogel solution to incubate. Non-perfused tissue will require longer incubation times, but no other alterations to the CLARITY protocol are needed.