Active Delipidation With SmartClear II Pro

Reagents / Equipment Required:

• SmartClear II Pro

• Delipidation Buffer – stored at RT

• Conduction Buffer – stored at RT

• SmartClear Membranes – v2 Required

4. Prepare the samples.

• Insert into an appropriately sized mesh bag. 

  • It is best to align the sample with the longest dimension vertically for best clearing speed (cerebellum down is best). Use the notches at the top of the mesh bags to identify samples. 

5. Load the samples into the device.

• Insert the mesh bag into the cylindrical sample holder and insert that in the clearing chamber. It is best to use the dividers to split the holder into quarters. 

6. Prepare the device for clearing.

• Tighten the knob on the clearing chamber and close the lid of the device. 

• Set the temperature accordingly. 

  • If your samples contain endogenous fluorescence: clear at 42°C for Buffer A (Delipidation Buffer). 

    • This is equivalent to the ‘Gentle’ setting in beginner mode. 

  • If your sample doesn’t have endogenous signal, you can increase the temperature of Buffer A to 50°C, or ‘Fast’ setting for faster clearing speed. 

[Note] If you want further control of temperature, operate the device in Expert Mode. For preservation of RNA for future FISH studies, clearing should be performed at 37°C. To operate at this temperature, you may need to reduce the current from 1500 mA to 1000 or 1200 mA. 

• Turn on Electrophoresis power and clear the samples. The samples will not appear transparent at this stage, and they will almost appear unchanged. 

• Set the clearing time. This is dependent on the sample.

  • Adult mouse brains will clear in 30 hours at 42°C. 

  • Rat brains will clear in roughly 7 days at 42°C. 

  • Smaller samples will clear faster. 

[Note]: Unique Tissue Types

If your sample is liver, we have found that an additional step will result in more transparent samples. Please follow the Liver Clearing Protocol to complete clearing of livers after Step 1 in the protocol above. 

If your sample contains significant connective tissue, adipose tissue, or skin, (such as whole mouse heads) we have found that an additional dehydrating clearing step helps clear these other tissue types without damaging fluorescent proteins. Please follow the fDisco protocol after completing Step 1 in the protocol above. Please note that this step has the potential to harm antigenicity. This step can also be moved to after immunolabeling if you find that to be the case. 

If your sample contains a lot of native pigment or heme, it is best to add some kind of bleaching or decolorizing step. Some tissues that may need this step include: pigmented eyes, skin, spleen, heart, embryos, samples that have not been perfused, and old human or NHP brain tissue containing lipofuscin. There are several options to address this problem.  You can incubate the tissue overnight in 5% H2O2 in PBS at 4°C (or until white), or make an LED photobleacher. These options will bleach out any native FP signal. It is possible that some decolorizing agents from CUBIC protocols may work, but we have not tested them extensively at LifeCanvas. 

7. Transfer to PBS with 0.02% sodium azide at the end of the experiment.

• Store the samples at 4ºC until you are ready for the next steps.

8. Wash and shut down the device.

At the end of delipidation, the samples will look the same as before clearing and should not have changed size. You will not be able to tell that the sample is done clearing until the index matching stage. See images below.

You have finished Delipidation and may now move on to the next step. If your sample requires labeling, move on to that section. If no labeling is required, you can move on directly to Index Matching.