I have some questions about 'Preparing Frozen Tissue Sections'. First, If I should immerse the sample into the O.C.T. and freeze the sample in to liquid nitrogen ? second, I want to detect a trans-membrane protein , which I can used to fix the sections, 4%PFA or Acetone? Finally, Is there anybody could give me a protocol about the frozen section.Thanks!
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#2
LostintheLab
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Posted 11 March 2010 - 11:37 PM
1. put your tissue into the OCT compound in the best orientation for cutting your sections later (so you try not to cut angled slices)
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.
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.
I knew it! I knew it! Well, not in the sense of having the slightest idea, but I knew there was something I didn't know.
#3
Rsm
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Posted 12 March 2010 - 01:34 AM
Very good advice, LostInTheLab-chan.
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.
Best,
Minna
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.
Best,
Minna
I got soul, but I'm not a soldier
#4
tantao
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Posted 14 March 2010 - 07:26 PM
Thanks a lot!LostInTheLab-chan and Minna. Yours Tao
