My current protocol involves 2-methylbutane and dry ice. I do everything I can to ensure all reagents are as cold as possible so that freezing time is below 1 minute. I store 2-methylbutane at -80 prior to snap freezing. I also store glass beakers that I place my molds on filled with 2-methylbutane and in dry ice in a styrofoam ice bucket with the lid closed at -80 so that they are very cold. Last, I store my molds with OCT on ice prior to arranging my sample (brain tissue) for freezing. I normally achieve freezing time beneath 1 min and then store at -20 because I cyrosection within 1 week. I also like to store at -20 because this matches the temperature or the cryostat's chamber.
My lab recently moved to a new location and my theory is that our -20 isn't holding temp or at least that my samples are not quite -20. When I then go to cryosection my samples perhaps the temperature difference results in poor slices. So I basically want a protocol not involving liquid nitrogen that consistently produces good slices. Thanks.
Hi William,
it does not sound like your freezing procedure is the problem, we do the same but are not quite as stringent.
We store our samples at -80°, especially for long term storage. Equlibrating them to -20° can either be done in the cryostat (what we do) or in a -20° freezer for maybe an hour.
Hi WIlliam,
Your freezing procedure seems good. Maybe you could try to cool down the dry ice temperature by adding 99Etoh just before your proceedure.
I also store my sample at -80°C before cutting and let them reaching the cryostat temperature at least for 1h before cutting.
Best
I always add dry ice to my methylbutane-- once it stops bubbling, it is cold and should achieve a snap freeze in a matter of seconds. Sounds like you are just fine on the freezing procedure as others above have mentioned.
Some tissues also require specific temps (muscle may be different than brain, for example) for ideal cutting. Check and make sure this isn't the case before jumping to conclusions on the cryostat temp.
Hi all,
Thanks for the responses. This is super helpful. I will check on the cutting temp for brain. I recently tested different temps for cutting so knowing if my conclusion matches what others think will be good too.
Vincent, are you saying to pour 99% EtOH over my dry ice before freezing? I want to make sure I'm following you correctly. Thanks.
Tré
Hi,
I use acetone, dry ice and 2-methylbutane for snap freezing my samples. I will have acetone in a styrofoam box, put some dry ice on acetone to cool down its temperature.Then, i will put a beaker with 2-methylbutane in acetone. I will freeze Cryostat mold with samples embedded in OCT, gelatin in 2-methylbutane. This works for me.
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