Revision as of 21:37, 15 April 2014

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.

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

ETC Set-up

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.

Sample does not look clear even after a week of ETC

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

Loss of fluorescence following ETC

Imaging

White deposits forming when the sample is in FocusClear

Autofluorescence is blocking the signal