Would passive clearing reduce tissue expansion compared to ETC?

xyang

Hello,

My sample is 1 mm-thick mouse brain slice, and I really want to avoid tissue expansion during the clearing process (even it finally shrink back to the original size in the RI matching solution). So I was wondering whether I should use ETC or passive clearing? Would passive clearing have less tissue expansion compared to ETC? Since my brain slice is relative thin, how long will passive clearing take? Any suggestion would be highly appreciated!

Jake

From my personal experience, expansion may be caused by osmotic difference by the clearing solution used. Even without electric current, tissue will be expanded in the clearing solution. The best way to reduce the expansion is reducing the overall time in the clearing solution. The commercial device mentioned in the previous post (X-CLARITY from Logos Bio) is a good alternative. Only few hours (1-2 hr usually) are sufficient to clear a 1-mm thick mouse brain slice. And as well noticed, the expanded tissue shrink back to near its original size after incubating in an RI matching solution such as FocusClear or X-CLARITY Mounting Solution.

xyang

Thank you so much for your suggestion, Jake!

Kristin_Engberg

Tissue expansion is not exactly related to the time spent in the clearing solution. It is true that the longer the sample sits in the clearing solution, the more expanded it gets, but this is only because the lipids are clearing out of the tissue and the hydrogel is expanding to its' desired equilibrium state. This will happen regardless of the clearing method - as lipids are removed from the sample, the hydrogel swells to a point of equilibrium. This equilibrium point is different in the imaging solution, which is why the sample shrinks back down in certain RI matching solutions (in contrast, the sample swells quite a lot when placed in glycerol for imaging).

Unfortunately, you have no control over the swelling based on the clearing method you choose. However, adjusting the hydrogel solution will alter the hydrogel network composition which may in turn affect how swollen your sample gets during clearing. To achieve less swelling, you want a hydrogel that is more dense and crosslinked, as crosslinks and entanglements prevent polymer swelling. This could be attained by increasing the concentration of the acrylamide and bis-acrylamide in the hydrogel solution (although I've never actually tried this with a CLARITY sample - I'm purely going off of general hydrogel principles). Unfortunately, the major downside to increasing acrylamide concentration and crosslinking is that the network will become less porous, meaning that it will take longer to clear the samples and to stain them. Since you are working with 1 mm thick tissues, this may not be a significant problem if you're really concerned about swelling, so you will have to weigh the cost benefits yourself.

Leo

As a matter of facts, higher conc. of Acrylamide generated more swelled results. My opinion is usging strong fixative material or higher conc.