Following sample clearing in the clearing solution, the hydrogel-embedded tissue is washed in PBST buffer to remove SDS micelles. If the sample was labelled with fluorescent proteins prior to CLARITY processing (from a transgenic mouse line, for example), the proteins will be attached to the hydrogel matrix, and the sample can be imaged immediately after the PBST washing step. Otherwise, the clarified samples can be labelled via immunostaining and then prepared for imaging.

Sample preparation

To prepare the hydrogel-embedded tissue sample for imaging, it is first removed from PBST solution and placed in a mounting solution. After reaching an equilibrium swelling state in the mounting solution, the sample is mounted in a chamber containing the same solution for imaging.

Place in mounting solution

Procedure

Remove the tissue sample from PBST where it was swollen for washing/staining/storage. Place the sample in a few milliliters of the mounting solution. Only enough solution to fully immerse the sample is needed (~4 mL for a whole mouse brain, for example). Incubate the sample with gentle shaking at either room temperature or 37°C for several hours or overnight. It is not recommended to leave the tissue sample in mounting solution for extended periods of time before imaging, but a day or two should be fine as long as the PBST washes were completed to rinse out residual SDS micelles from the sample.

Mount in imaging chamber

Once the tissue appears visually transparent and has de-swelled back to its anatomical size, the sample is ready for mounting. A sealed imaging chamber is constructed to hold the sample and mounting solution.

Imaging techniques

Imaging should be performed using a microscopy technique that focuses on one plane within the sample at a time. Standard light microscopy will not be suitable for the optically transparent, intact tissue samples as light will penetrate the entire sample and fluorescence will be emitted from everywhere. Thus, the signal from the focused plane will be diluted and probably lost by the signal coming from everywhere else in the sample, causing the sample to look like a blob of autofluorescence under the light microscope.

Confocal microscopy

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Light sheet microscopy

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