[Histonet] Frozen tissue detachment

[Marie Madsen]

Hi everyone,

We're having trouble with frozen tissue detachment (mouse aortic root).
Our procedure is as follows:

1. Fresh heart into NBF 4% (Lilly's) 24h, fridge (+4)
2. OCT (tissue-tek) 2h, fridge (+4)
3. Quickly frozen in icecold isopentan
4. Freezer (-20) until sectioned at 10 µm in cryostat (at approx -20)
5. RT one to several hours before being put back into the freezer.

We use Superfrost glass slides.

The *only* thing I can think of is wrong, is that the sections have been thawed/frozen several times before being used for staining (we do that to find the best section for our stainings) -do you think that's the problem?

Another thing could be that after being sectioned we don't have a specific amount of time and temperature at which the sections should dry..

I found out recently regarding my IHC protocol, that removing the OCT with 70% EtOH (10 min => dry 20 min) makes the tissue stick better than removing it with dH2O or TBS..but then the specific staining also became much more faded..any ideas what else to do?

Sorry about all the questions -I'm relatively new to this field, but find it very interesting.

Best, Marie