Tissues should be fixed for as little time as necessary before being moved to a storage medium (e.g., 70% ethanol) to prevent over-fixation and subsequent difficulties during sectioning. I am not aware of any methods that can be used to reverse the over-fixation.

I doubt that fixation causes excessive hardening. I guess the specimens are a few millimeters thick. How long stays the tissue in absolute ethanol and xylene? Temperature?

When you put the block with the surface in water for 5 min, does the tissue turn white and swell?

Then it is overprocessed and over-dehydrated. After rehydrating the surface cutting should be better.

Gudrun Lang

First of all, thank you very much for your reply.

I need a lot of advice on this very difficult process..!

To answer, the thickness of the fixed tissue was about 10mm.

After fixation in 4% PFA for 24-36 hours,

I washed it in running water overnight.

I refrigerated it in 70% ethanol until further analysis.

During the processing, I gradually dehydrated it using a machine at room temperature, and after 3 hours in absolute ethanol and 3 hours in xylen, I overnighted it in 60°C paraffin.

As you mentioned, when I soaked the tissue block in water for 5 minutes, it did not turn white or swell.

If it was due to excessive processing and dehydration, is there an effective way to rehydrate it?

I would appreciate it if you could reply.

John Hardy Lockhart

Thank you for your reply. If I find a good solution, I will share it with you.

If over-dehydrated you can try to soak the block in 10% citric acid. But be careful not to soak it too long because of swelling.

Do all kinds of tissue behave the same way? I think, cutting of fat tissue should improve with long incubation in xylen and paraffin.

Muscle is full of collagen, that would tend to shrink and harden in the hot paraffin. Liver renders brittle and crumbly.

There should be some experts for animal tissue out there.

In my opinion you could skip the water rinse and go directly into the 70%.