SmartClear II Pro Mouse Brain Clearing Tips

1. First determine if you need to clear a whole mouse brain. If your experiment allows it, even slicing the brains into hemispheres can drastically increase clearing speed and any subsequent immunolabeling.


2. Next, you should determine what clearing temperature you wish to use. We have found that 42°C is optimal for clearing samples with endogenous fluorescence and using the aqueous-based tissue-clearing technique. This is the default temperature of the “Gentle” setting in Beginner Mode. If you do not have any fluorescence, you can increase the temperature of Buffer A to 50°C to increase clearing speed.


3. If you are worried about the temperature of Buffer B and the set temperature, don’t worry! Only Buffer A is in contact with the sample, so the temperature of Buffer B is not critical.The temperature of Buffer B is not as highly regulated and some variability is expected.


4. When loading the samples into the clearing chamber, always try to align the sample with the long axis oriented vertically. This will ensure the shortest distance is parallel to the electric field, which maximizes clearing speed. To get samples to “stand up” vertically, you can use some nylon mesh and roll it into a small cylinder and place it in the sample cup with the sample to hold it in place.

Mouse Hemisphere

Long axis oriented vertically
A small (~4 cm x 3 cm) piece of nylon mesh rolled into a tube and inserted into the sample holder can help orient a sample vertically.

5. The progression of clearing is outside-to-in. If you see an opaque core, then clearing should continue until the sample looks evenly translucent. After index matching in EasyIndex the sample will become transparent.

Clearing Progression

6. The time for the 10-day lifetime of the buffer and membranes is accumulated when the clearing power is turned on. If you anticipate some downtime of a day or two between samples, you can simply leave the membranes and buffer installed, turn off the clearing power, and keep the pumps running during this downtime. This will ensure the membranes stay hydrated.


7. If you do not anticipate clearing more samples for more than a few days and you have not yet reached the 10-day lifetime of the buffers and membranes, you can save them for later. Simply drain the buffers into separate containers (one for A and another for B), and place the membranes in a sealed container with DI water or PBS. You can reinstall the membranes and buffers later when you want to start clearing again.


8. In general, it is good to re-calibrate the temperature sensors every few months and when you receive a new system. We do calibrate the systems in house, but the calibration is dependent on the ambient temperature. This will ensure your buffers are the optimal temperature. See the owner’s manual for detailed instructions.