Working with some human spinal cord. I took this fresh and embedded in OCT, and then froze in isopentane cooled by liquid nitrogen. We then stored in an airtight container at -80C before sectioning.
These are the sections we got
What’s the diagnosis?
- Freezing was too slow
- The white matter looks OK. This is a fairly large piece of tissue. Did the interior not freeze quickly enough and ice crystals formed and damaged the tissue?
- Too cold at cryostat
- We sectioned at -20C and then even brought it down to -13C. So I don’t think it was too cold, but I know tearing can happen with too cold blocks.
- 10x discusses this for the Visium protocol:
- Something else I’m not thinking about?
I’m inclined to think maybe not fast enough freezing. In which case, directly freezing the tissue in isopentane (not in OCT), might be the way to go.