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I would like to do a quick freeze on pancreas from mice. I want to then make sections (30 µm thick). The idea is to preserve a fluorescent staining done in vivo.

I do not know if I should place my fresh tissue directly into liquid nitrogen, then embed the tissue in OCT, and then freeze everything in cold isopentane?

Or do I have to embed the fresh tissue in OCT first, prior to putting everything in nitrogen?

Or is there another protocol to do quick freezing in order to do sectioning?

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2 Answers 2

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Embed the tissue in OCT first (roughly cut to remove everything what is not needed) and the flash freeze it. Then you can cut your sections.

The first protocol describes it in detail, the second gives some additional information:

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do not place it directly in the N2 unless it's clean. and high grade liquid N2 costs an arm and leg. put the tissue in the oct in a conical vial and that in the N2. don't use polycarbonate it may crack. check with the OCT manufacturers suggestions first though. I've followed this BD protocol before

http://www.bdbiosciences.com/resources/protocols/frozen_tissue.jsp

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