Saponin

jdscott

Has anyone used saponin for hydrogel embedding of non-perfused tissues? Has it made a difference in the amount of time needed for the hydrogel to diffuse into the tissue? Have you had problems with bubbles in the tissue? I am clearing mouse livers, and I've been able to clear pretty well, but I would like to start using less hydrogel solution if possible by eliminating the perfusion step. Thanks!

DominicJG

Hi jdcott,

I haven't used saponin yet. However, I'm going to compare w/ and w/o saponin in the next couple of days. Will let you know about my experience. Switching gears, do you mind to post one of your mouse liver cleared tissues?

Thx

jdscott

Are you also clearing livers? I tried clearing without perfusing with hydrogel (I incubated the liver in hydrogel with saponin for several days). Here are some pictures of what I've been able to get so far.

The piece on the right in this picture is embedded, uncleared liver. The piece on the left was cleared for four weeks. This size seems to be too large to clear effectively. https://drive.google.com/file/d/0B4-Nd_LhtJMgR1FtaXUtWTVJdlU/edit?usp=sharing

This is another piece of the same liver, non-perfused but embedded with hydrogel using saponin. It has been going for about four weeks as well. https://drive.google.com/file/d/0B4-Nd_LhtJMgdUEtWXJlS0dFWTA/edit?usp=sharing

Here is a bit of liver that was perfused with hydrogel and cleared for two weeks. The one on the left is uncleared for comparison. https://drive.google.com/file/d/0B4-Nd_LhtJMgUExnaHBEcXg5U0E/edit?usp=sharing

From my experiments, I would say hydrogel perfusion is necessary - otherwise it takes too long and doesn't clear quite as well. I have not tried using saponin after doing the hydrogel perfusion. I'm very interested to hear your results!

KeithSiew

We're going to attempt using saponin to help with some post-mortem human tissues. Have you any further updates on your experience with saponin?

jdscott

At least for liver, saponin didn't make a noticeable difference. My goal in using saponin was to see if I could achieve deeper infusion of hydrogel monomer if the tissue itself was not perfused. I'm assuming you have similar goals if you are using human tissue. I found that hydrogel monomer was not able to diffuse into non-perfused tissues very well, regardless of presence of saponin, even at longer incubation times (up to 10 days). This may very well depend on tissue type, however. Another thread on these forums mentioned proteinase K treatments and freeze-thaw treatments. I am considering using these methods to improve antibody penetration, but they might also improve hydrogel monomer penetration. I'm not sure how well non-fixed tissues would hold up to those treatments, though. Good luck!

Vortexam

Hello Scott,I am just starting clarity method for clearing Liver.I have another question,I am having roube in making hydrogel solution.It is polymerizing well but has certin small air bubbles.I am not using saponin as it is metioned in the protocol that use of saponin is optional.Is it possible to cut down this problem or any other solution.For how long did you added Nitrogen gas in the chamber? I have my own little dessication chamber and I am wondering if the bubbles are due to higher vaccum or nirogen pressure.