I have been working on cryosectioning adult zebrafish with large tumors in their abdominal region, but I am getting a significant degree of holes/tearing in the sections as they come off the block. I'm not able to fix the fish before sectioning as the experiment the sections will be used for requires fresh-frozen tissue.
My method: I am euthanizing the fish in ice water and then immediately flash freezing in OCT/dry ice/ethanol. I am leaving the blocks at -80 for at least 24 hours before sectioning, which seems to help with the tearing issue somewhat.
There is not much information on cryosectioning zebrafish online (that I could find) so I was wondering if anyone has any tips?
you used OCT/dry ice/ethanol. What is the purpose of ethanol here?
Second when you take out blocks from -80C, do not start cutting right away, equilibrate blocks to Cryostat temperature so that blocks comes to -20 to -22C, than start cutting, that might help. As Mohammed Mahdy mentioned the same.
Hi,
Please, have a look at these two documents:
https://www.frdc.com.au/-/media/Fish-FRDC/Environment/Animial-Health-and-Biosecurity/Humane-Euthanasia-Techniques-for-Ornamental-Fish-AAWS-Document.ashx?la=en
https://www.leicabiosystems.com/pathologyleaders/freezing-biological-samples/
Also, did you coat the mold (sitting in some dry ice) with an OCT layer prior to covering the zebrafish with OCT completely and keeping it on dry ice?
You might have some ice crystals forming if you place the freshly eutanized fish directly in the mold with OCT on dry ice, since freezing is a bit slower.
If the above issues have been avoided, have you considered mucin as another possible culprit:
https://academic.oup.com/mbe/article/33/8/1921/2578619
Article Mucus as a Barrier to Drug Delivery - Understanding and Mimi...
Article Barrier properties of mucus
as mucin expression pattern and composition is altered in cancer animals.
I hope this helps :)
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