I have perfused mice brains with paraformaldehyde then processed them to 40% sucrose during a short visit abroad, hoping to be able to take them with me in the end of my visit. However, I discovered sending refrigerated/frozen samples to Brazil is extremely hard as our boarder takes an impossibly long time to process packages, even from courier services such as DHL and FedEx.
I was wondering if anyone have ever returned sucrose-protected brains to paraformaldehyde and re-processed them to 70% alcohol. It would limit my options, but at least I would be able to post them in unrefrigerated packages. I appreciate anyone's experience with this!
Dear Isis,
If you want to dehydrate in alcohol , you will need to remove the sucrose slowly--basically the reverse of sinking them in sucrose solutions. Then, rinse well with PBS before returning to fixative. However, it might be easier to transfer them to a paraformaldehyde+sucrose solution, unless you are worried about shipping the fixative solution. It will depend on what your ultimate use of the brains will be. One lab in which I worked routinely used paraformaldehyde + sucrose to cryoprotect the rat brains that were sectioned on the freezing microtome. Those sections were collected in wells of PBS, so both the extra paraformaldehyde and sucrose were diluted before performing routine Nissl or silver staining.
Good Luck!
Jill
Dear Jill Miotke , thank you for your answer!
Wouldn't the paraformaldehyde+sucrose solution require the brains to be kept refrigerated as well?
Dear Isis de Oliveira,
It would be better if the solution was kept at 4 C, but my thinking was that the added paraformaldehyde could give you a bigger window of preservation if the samples had to sit once the cold packs reached room temperature. I have sunk tissue on a rotator at room temperature when I was desperate to process samples, so a short time in sucrose outside of 4 C is possible. It just depends on your end use of the tissue, and the time it will take for the shipment to be delivered.
Good Luck!
Jill
You can do it, remove sucrose slowly in gradient way, in PBS, in 10%Neutral bufferred formalin or 4%PFA, PBS, 70% ethanol