Two alternatives to the nitrogen purge for the hydrogel

Genesprite

I attended the CLARITY workshop, and while there, Kristin mentioned that someone was using Peanut oil on top of the hydrogel solution, or filling the tube to the top, to prevent oxygen from inhibiting the polymerization. These sounded like good alternatives to the nitrogen purge. The main problem with filling a 50 ml conical tube to the top with hydrogel, is the waste of reagent.
The first thing I tried was transferring the brains to 15 ml conical tubes and filling them all the way up to the tippy-top before closing the tube and placing in a 37 degree water bath. This worked great!! It isn't too hard to remove the mouse brains from the 15 ml conical, and it uses less hydrogel. I did have to use a metal spatula to get it lose, but it was fine. I also recently tried adding sunflower oil to the top. I didn't have peanut oil, but I did have sunflower oil on hand, it also worked great! This saves the space and cost of nitrogen tanks, vacuum pumps and desiccator. Definitely a big thumbs up.

jianbo

It really saves a lot of time and works fine!

rob

I agree. Topping up your tube of hydrogel with oil ( I have used a mixture of different oils, including sunflower oil) works and is quick and easy

coweight

Do you vacuum before adding oil?

Adam

Just add oil before polymerisation, and it should work fine. I usually find whereas with degassing, polymerisation takes about 60-90 minutes, with oil instead it takes up to 2 hours, but it works.

jose_jpr

Is there a protocol of this technique? thanks

Adam

You don't really need a protocol, just open the tube, put some oil on top, then put in a water bath until the hydrogel polymerises.

BJ2014

After reading this thread, I tried pouring a 4% Acrylamide (diluted from 40% stock solution, Fisher BP1410-1) supplemented with .25% photoinitiator into a 15mL Falcon tube. I poured the cold solution until it filled the tube, such that no air was in it when I screwed the cap on. However, I saw within 30min into the 37degC water bath incubation that small bubbles started forming along the sides of the tube, but this did not turn out to be problematic. In the end, the gel formed nicely, and my tissue is intact after 8% SDS clearing in 37degC incubation chamber. If you guys see bubbles along the tube walls, don't freak out. The gel will still form.