Clearing Solution issues

SD07

I noticed in the brains I've cleared there has been a white precipitate forming when placed in Focus Clear, and when viewed under the microscope there are very noticeable holes in the tissue. We think it may be the result of the way we made the clearing solution. When making the solution, we just added the boric acid and sds, followed by the dH2O. A white precipitate formed, and it took more than 200 pellets of NaOH (for 10L of clearing solution) to bring the pH to 8.5.

Has anyone had this issue, or is there a specific protocol that works better for making the clearing solution (like dissolving either boric acid or sds first?). Thanks!

lijun

What's the size of the holes you've observed ? Is it as big as the nucleus? I found holes in my brain sections too,and then I did a DAPI staing.Almost every hole has a nucleus inside.

Fabio

Hi I have never seen a precipitate when mixing the solution.
I will recommend first prepare the boric acid and adjust pH, when the pH is
set to 8.5 add the SDS. At the end check the pH again with some pH paper

Kristin_Engberg

I am not sure that the clearing solution would cause holes in your tissue unless there was something wrong with the hydrogel embedding step (and then the clearing solution would clear out unbound molecules no matter how it was made).

Still, I agree with Fabio's method to prepare the clearing solution. I usually recommend preparing a 2L solution of boric acid in water and adjusting the pH to 8.5 (you will probably need to start adding NaOH to get the boric acid to dissolve completely). You can then add the 2L solution to a 10L container containing the SDS and add the necessary volume of water. Check the pH, but it should remain stable at 8.5.

SD07

Awesome, I'll try that--thanks for the suggestions! lijun - the holes were fairly large. Much bigger than a nucleus.

Kristin_Engberg

@SD07 - how long did you have your sample in FocusClear before imaging? The development of a precipitate and "holes" under the microscope sounds like what happens after the sample has been in FocusClear for too long. We have noticed that after a few days in FocusClear, a precipitate starts forming throughout the sample, causing those areas to be blacked out when imaging.

Instead of storing our samples in FocusClear, we keep them in PBS until the day before we are ready to image, and then place them in FocusClear.

SD07

@Kristin_Engberg We had our sample in FocusClear for 2 days. Perhaps that's the issue--we will try leaving it in for a shorter period with the next samples. Thanks!!

Shashank20

Hi I am working at Lund University Sweden and Copenhagen Univ. Denmark and would be interested to know :

1) A distributor of Focus Clear in Europe. Apparently it looks like cell explorer labs is from taiwan. Any suggestions for focus clear distributors in Denmark?

2) Does anybody has a experience of imaging the ETC cleared tissue under BABB (Murray's Clear) ?

Thanks!!