How to embed already frozen tissues in OCT

Hi all,
We have some spinal cord samples that were snap frozen in isopentane at the time of harvest. They’ve been in the -80 ever since.

I want to embed them in OCT for cryosectioning. What is the best way to do this so as to maintain the best morphology?

@tberta @liz

Hi Alex,

Just saw this here. I regularly snap freeze spinal cord samples and store in -80 before sectioning. When I want to section I place the frozen sample in the croystat (usually it’s at -20 to -25 degrees C) and let it adjust to new temp for about 30 minutes. Then I embed in OCT by using a mold such as pipette tip with end cut off. Place pipette tip on one of the chucks and place the spinal cord sample vertically into the pipette and fill it with OCT. Usually works pretty well for me and morphology seem fine

That’s great! So you bring it down to the right temp, then mount. I’ve heard that the OCT doesn’t stick as well to the sides of already frozen tissue. I’ll give it a try. Thanks!

@DaraBree How do you get the pipette tip off once the OCT freezes?

I make a cut in the pipette tip from vertically top to bottom using a backed blade. You should then be able to essentially peel off the pipette tip off form the frozen OCT. A forceps helps if you are having trouble doing it with your fingers. Try to do it in the cryostat to help keep the OCT frozen

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You can use a bath of Isopenthane, cool it down to -50/-70 C using liquid nitrogen (do not add it directly to the Isopenthane). Have some tissue molds, containing your brains, on dry ice, and when the bath has reached the right temperature, very quickly place OCT on top and dip them in the Isopenthane. If you’re quick enough your tissue should not have too much of a heat shock.

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