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CLARITY is a multi-step process, and sometimes it can be a little tricky to get all the steps working correctly, especially when starting out. Here are some common problems and concerns that may be encountered along with suggestions for troubleshooting.


Sample Preparation

What to do with tissue samples that can not be perfused with hydrogel solution

To prepare tissue samples that can not be perfused with hydrogel solution for CLARITY, simply place them in the hydrogel solution for incubation. The samples will require slightly longer incubation times than samples that have been perfused, but otherwise they undergo identical clearing and processing steps.

Hydrogel Polymerization

After 3 hours of polymerization at 37°C, the solution is still liquid (no gel is formed)

This could be caused by too much oxygen present in the sample container during polymerization (unsuccessful degassing) or improper control of the temperature. See the troubleshooting section in Hydrogel Embedding.

Trouble removing the gel from the tissue sample without causing damage

This could be a problem if the tissue sample is quite small or fragile. If this is the case, it is probably best to remove the bis-acrylamide from the hydrogel solution, such that the solution around the tissue sample will not form a gel.

If the gel surrounding the tissue is stiff and rigid, such that its removal tears the tissue, the polymerization during high temperature incubation was likely performed for too long. Make sure to incubate for only about 3 hours, or until a soft gel is formed.

Sample Clearing

Sample is swelling in clearing solution

Sample swelling in clearing solution as the tissue clears is normal and expected. This happens because the lipids are leaving the tissue and no longer restraining the polymer chains making up the hydrogel from swelling to a larger size. The sample will return to anatomical size when swollen in the mounting solution prior to imaging.

Trouble getting clear tissue samples with ETC

Try setting aside a few samples for passive clearing when setting up ETC for the first time. If these samples do not clear slowly over time, there may be a problem in the sample preparation or hydrogel embedding steps. For instance, if the sample is left under high temperature incubation for too long, the hydrogel network may not be porous enough to support clearing.

Alternatively, if the passively cleared samples slowly clear as expected, there could be a problem in the ETC set-up or settings. Also be aware that samples may not look transparent immediately after ETC clearing and may need a couple days of passive clearing to remove the residual lipids from the tissue. Along those lines, if ETC alone is not working well, try an alternative clearing strategy, such as a combination of passive clearing and ETC.

Sample turns brown after running ETC

The tissue sample should not turn brown during ETC. This is an indication of damage to/melting of the tissue. Throughout ETC, the tissue should instead remain a whitish to pale yellow color. Most likely, the damage to the sample is from the temperature inside the chamber getting too hot. This can be fixed by increasing the flow rate of the circulating clearing solution and by lowering either the set temperature or voltage (or both). For more information on ETC settings, see ETC set-up.

Black residue is forming on the platinum electrodes and depositing on the sample

A black residue resulting from the electrophoresis process does build up on the platinum electrodes over time. To prevent the residue from depositing on the sample, the electrodes need to be cleaned every two to three days of use.


White deposits forming when the sample is in FocusClear

An irreversible white precipitate can form after the sample sits in FocusClear for a couple days. These white deposits are usually caused from residual SDS micelles left in the tissue sample. Be sure to thoroughly wash the sample in PBST for at least 2 days before transferring it to the FocusClear solution.