Hydrogel Solution Not Working

DRTMCL

Hello All,
My lab is relatively new to CLARITY work. About 6 months ago, we were able to get a solid batch of embedding to work, to the point that we were successfully embedding tissue and passively clearing them. We followed the CLARITY wiki's embedding and clearing directions to the T. The first time, we made all of our own hydrogel solutions and found this success. Then about 3 months ago we were no longer able to get the polymerization to occur. The initial batch just stopped working. Since then, we have tried remaking solutions, improving degassing, buying standard stocks, monitoring pH, using replacements (APS instead of thermal initiator, fresh BIS, fresh Acrylamide) to try and nail down what exactly was causing the polymerization to fail, only to be left scratching our heads and unable to replicate. Turning to you guys, do you have any recommendations? Has anyone encountered similar conditions? Any guesses at all will probably help us solve this puzzle. Thank you.

Kristin_Engberg

That's very puzzling. The polymerization should be consistent, especially with a newly-made batch of solution. Did you happen to lower the amount of acrylamide (i.e. less than 4% concentration)? Lower concentrations will not usually form a complete hydrogel in solution due to lack of crosslinkers (this is not a concern in the tissue however due to all the proteins/biomolecules that crosslink the polyacrylamide together). My only other thought would be that perhaps the thermal initiator has expired or gone bad. Was it stored in a fridge?

DRTMCL

Thanks for the reply Kristin! We ended up ordering the stock 16% PFA and that did not work. We also ended up ordering a new thermal initiator and that also has not yielded successful results. I will say, that there did appear to be some literature advocating for freezing the hydrogel solution or aliquoting the VA-44 and then freezing the initiator. We did not do this, just stored everything in a 4 degree cold room.