Rat brains won't sink in 30% sucrose?

[nzkat]

Rat brains were perfused using 4% para, then cryoprotected in 30% sucrose in 0.1M PB until they sunk (about 5 days).
They were then stored at -20 C in  cryoproteectant solution containing ethelyene glycol, 30% sucrose in 0.1M PB for the last few months. Now I want to cut them on our microtome so I stuck them back into the 30% sucrose solution to get out the antifreeze, but they are floating again.. are they safe to freeze??!

Thanks!

[excalibur]

You may need a larger volume of sucrose or more than one change.

[nzkat]

Hey - thanks, they have all sunk now, just took them a few days longer than expected. Must just take longer than one night to get the ethlyene glycol out and the surcose back in..

Cheers

[richard03]

Next time you may want to consider an intermediate sucrose (15%) between fixation and 30% sucrose. It should work better than putting fixed brains directly into 30% sucrose.