Hi all,
our lab is currently working on H&E staining with frozen liver section.
As you may see, the image shows that our tissue section has multiple large cracks.
When the liver was dissected, the tissue was washed with PBS and then fixed with 4% PFA overnight. Aftern then, the tissue was stored in 30% sucrose solution for dehydration. Dehydrated tissue was frozen with OCT compound and then cut in 15 um section.
Some said fixation must be avoided for frozen section but others disagreed. Is there anybody who can help us to make our image look better?
Best,
Hyeyoon
Thank you for your answer! I will try cryostat again without dehydration process and see how it goes :)
In the figure of the liver I don't see any nuclei. The tissue doesn't seem to be prevented in good quality. I would try to fix thin slices of liver (4 mm) in 4% NBF for one-two days, then go into your cryoprotection with sucrose, then make a 1:1 solution with OCT to infiltrate the tissue before freezing. Freezing should be fast but not too cold to prevent cracking. In the gas phase of liquid nitrogen or on dry ice at 70°C. Cutting should be done at about -20°C, so let the tissue get "warm" enough again before cutting.
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