Preparing Frozen Tissue Sections
Posted 11 March 2010 - 07:04 PM
Posted 11 March 2010 - 11:37 PM
2. You will need some isopentane in a freeze-proof container that sits in a resevoir of liquid Nitrogen, don't put your section directly in liquid nitrogen as it freezes to quickly destroying tissue integrity.
3. if you have space in your container, but your section block on a foil boat on the surface of your liquid nitrogen cooled isopentane or hold at the surface with tongs, make sure you don't get isopentane in your section as you will get ice formation and destruction of tissue.
4. Watch as the clear solution turns white, wait till completely white (a little dome appears in the middle of the block), you can now remove and store in -80C
If you are looking at a membrane protein then use PFA, acetone dehydrates and extracts lipids, so your membranes will be destroyed by acetone addition and disrupt your membrane protein.
Have a look under the protocol section of bioforum - top menu bar, second tab along. There should be some frozen section protocols there.
Posted 12 March 2010 - 01:34 AM
I would like to suggest a slightly different way of freezing the stuff: Pour liquid Nitrogen into a Styrofoam container. Slowly add a plastic dish (for example the lid of a 10cm dish), so that it floats on the Nitrogen. Then put your sample in OCT compound directly onto the dish, wait 3mins, et voila. No need of Isopentane, the sample freezes not too quickly (which is the case if you'd use a metal dish), and almost fail-proof. Just make sure your dish does not crack when you put it into the Nitrogen.