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!
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.