Table 3

CLARITY troubleshooting

The tissue is not clearingClosely monitor the pH of the clearing solution to ensure it remains between 8 and 8.5. The pH of the solution will decrease over time and solution may needto be replaced during the clearing process. Small volume recirculators will need to be changed more frequently than those that hold a large volume of SDS.
The tissue is “melting”High temperatures for a prolonged period of time may lead to melting. Ensure that the clearing solution temperature is appropriate. It may be necessary to increase the flow rate of the recirculator to maintain a suitable temperature in the chambers.
The tissue is turning dark yellow or orangeThis may be due to high clearing temperature (see above). A localized burning/color change may be the result of the tissue directly contacting an electrode. Tissue should be in close apposition to, but not directly touching, the electrodes. A plastic mesh divider should be placed between the tissue and electrode to prevent direct contact.
The brain has a black residue on the surfaceEnsure the cleanliness of the recirculator and filter. A large buildup of black residue on the platinum electrodes can be cleaned by reversing the polarity of the electrodes and running the ETC chambers for several hours. Always ensure that as much hydrogel as possible is removed from the surface of the tissue before beginning the clearing procedure as the hydrogel has a greater tendency to gather residue than the tissue itself.
Cleared tissue is turning opaque in glycerolOn occasion, tissue may become opaque following transfer into 80% glycerol. This can be corrected by returning the tissue back to PBST at 50°C until clear, typicallya few hours. The tissue can then be transferred back to glycerol. This issue seems to be the result of incomplete tissue clearing.
An opaque precipitate is forming in the tissue in FocusClearA seemingly irreversible black precipitate may begin to form in the tissue if it is left for prolonged periods of time in FocusClear. Tissue should not be stored in FocusClear any longer than necessary to equilibrate and image the tissue.